Mutant phosphoenolpyruvate carboxylase, its gene, and production method of amino acid

ABSTRACT

A phosphoenolpyruvate carboxylase gene, which has mutation such as mutation to replace 625th glutamic acid from the N-terminus of phosphoenolpyruvate carboxylase with lysine, mutation to replace 438th arginine from the N-terminus with cysteine and the like, is introduced into Escherichia coli or coryneform bacteria, so as to produce a phosphoenolpyruvate carboxylase which is not substantially inhibited by aspartic acid, thereby amino acid is efficiently produced.

TECHNICAL FIELD

The present invention relates to a mutant phosphoenolpyruvatecarboxylase, a gene coding for it, and a production method of an aminoacid, and in particular relates to a gene having mutation to desensitizefeedback inhibition by aspartic acid, and utilization thereof.

BACKGROUND ART

Phosphoenolpyruvate carboxylase is an enzyme which is found in almostall bacteria and all plants. The role of this enzyme resides inbiosynthesis of aspartic acid and glutamic acid, and supply of C4dicarboxylic acid to the citric acid cycle for maintaining its turnover.However, in the fermentative production of an amino acid using amicroorganisms, there have been few reports on effects of this enzymehas not been made clear (Atsushi Yokota and Isamu Shiio, Agric. Biol.Chem., 52, 455-463 (1988), Josef Cremer et al., Appl. Environ.Microbiol., 57, 1746-1752 (1991), Petra, G. Peters-Weintisch, FEMSMicrobiol. Letters, 112, 269-274 (1993)).

By the way, the amino acid is a compound which universally exists incells as components of proteins, however, for the sake of economicenergy metabolism and substance metabolism, its production is strictlycontrolled. This control is principally feedback control, in which thefinal product of a metabolic pathway inhibits the activity of an enzymewhich catalyzes the earlier step of the pathway. Phosphoenolpyruvatecarboxylase also undergoes various regulations in expression of itsactivity.

For example, in the case of phosphoenolpyruvate carboxylase ofmicroorganisms belonging to the genus Corynebacterium or the genusEscherichia, the activity is inhibited by aspartic acid. Therefore, theaforementioned amino acid biosynthesis, in which phosphoenolpyruvatecarboxylase participates, is also inhibited by aspartic acid.

In the prior art, various techniques have been developed for efficientproduction in amino acid fermentation, and fermentative production hasbeen carried out for leucine, isoleucine, tryptophan, phenylalanine andthe like by using mutant strains converted to be insensitive to feedbackcontrol. However, there has been known neither mutantphosphoenolpyruvate carboxylase converted to be insensitive toinhibition by aspartic acid, nor attempt to utilize it for fermentativeproduction of amino acids of the aspartic acid family and the glutamicacid family.

On the other hand, ppc gene, which is a gene coding forphosphoenolpyruvate carboxylase of Escherichia coli, has been alreadycloned, and also determined for its nucleotide sequence (Fujita, N.,Miwa, T., Ishijima, S., Izui, K. and Katsuki, H., J. Biochem., 95,909-916 (1984)). However, there is no report of a mutant in whichinhibition by aspartic acid is desensitized.

The present invention has been made from the aforementioned viewpoint,an object of which is to provide a mutant phosphoenolpyruvatecarboxylase with substantially desensitized feedback inhibition byaspartic acid, a gene conding for it, and a utilization method thereof.

DISCLOSURE OF THE INVENTION

As a result of diligent investigation in order to achieve theaforementioned object, the present inventors have found that theinhibition by aspartic acid is substantially desensitized by replacingan amino acid at a specified site of phosphoenolpyruvate carboxylase ofEscherichia coli with another amino acid, succeeded in obtaining a genecoding for such a mutant enzyme, and arrived at completion of thepresent invention.

Namely, the present invention lies in a mutant phosphoenolpyruvatecarboxylase, which originates from a microorganism belonging to thegenus Escherichia, and has mutation to desensitize feedback inhibitionby aspartic acid, and a DNA sequence coding for the mutantphosphoenolpyruvate carboxylase.

The present invention further provides microorganisms belonging to thegenus Escherichia or coryneform bacteria harboring the DNA fragment, anda method of producing an amino acid wherein any of these microorganismsis cultivated in a preferable medium, and the amino acid selected fromL-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid,L-arginine and L-proline is separated from the medium.

Incidentally, in this specification, the DNA sequence coding for themutant phosphoenolpyruvate carboxylase, or a DNA sequence containing apromoter in addition thereto is occasionally merely referred to as "DNAsequence of the present invention", "mutant gene" or"phosphoenolpyruvate carboxylase gene."

The present invention will be explained in detail hereinafter.

<1> Mutant phosphoenolpyruvate carboxylase

The mutant phosphoenolpyruvate carboxylase of the present invention(hereinafter simply referred to as "mutant enzyme") lies in thephosphoenolpyruvate carboxylase of the microorganism belonging to thegenus Escherichia, which has mutation to desensitize the feedbackinhibition by aspartic acid.

Such mutation may be any one provided that the aforementioned feedbackinhibition is substantially desensitized without losing the enzymeactivity of the phosphoenolpyruvate carboxylase, for which there may beexemplified mutation which, when a mutant phosphoenolpyruvatecarboxylase having the mutation is allowed to exist in cells of amicroorganism belonging to the genus Escherichia, gives the cellsresistance to a compound having the following properties:

it exhibits a growth inhibitory action against a microorganism belongingto the genus Escherichia which produces a wild type phosphoenolpyruvatecarboxylase;

the aforementioned growth inhibitory action is recovered by existence ofL-glutamic acid or L-aspartic acid; and

it inhibits wild type phosphoenolpyruvic carboxylase activity.

More concretely, there may be exemplified, as counted from theN-terminus of the phosphoenolpyruvate carboxylase:

(1) mutation to replace 625th glutamic acid with lysine;

(2) mutation to replace 222th arginine with histidine and 223th glutamicacid with lysine, respectively;

(3) mutation to replace 288th serine with phenylalanine, 289th glutamicacid with lysine, 551th methionine with isoleucine and 804th glutamicacid with lysine, respectively;

(4) mutation to replace 867th alanine with threonine;

(5) mutation to replace 438th arginine with cysteine; and

(6) mutation to replace 620th lysine with serine.

Incidentally, as the phosphoenolpyruvate carboxylase of themicroorganism belonging to the genus Escherichia, an amino acidsequence, which is deduced from a phosphoenolpyruvate carboxylase geneof Escherichia coli (Fujita, N., Miwa, T., Ishijima, S., Izui, K. andKatsuki, H., J. Biochem., 95, 909-916 (1984)), is shown in SEQ ID NO:2in the Sequence listing. In addition, an entire nucleotide sequence of aplasmid pT2, which contains the phosphoenolpyruvate carboxylase gene ofEscherichia coli, is shown in SEQ ID NO:1 together with the amino acidsequence.

The aforementioned mutant enzymes are encoded by DNA sequences of thepresent invention described below, which are produced by expressing theDNA sequences in Escherichia coli and the like.

<2> DNA sequence of the present invention and microorganisms harboringthe same

The DNA sequence of the present invention is DNA sequences coding forthe aforementioned mutant enzymes, and has mutation to desensitizefeedback inhibition of phosphoenolpyruvate carboxylase by aspartic acidin coding regions in DNA fragments coding for phosphoenolpyruvatecarboxylase of the microorganism belonging to the genus Escherichia.

Concretely, there may be exemplified a DNA Sequence coding for thephosphoenolpyruvate carboxylase having the mutation of any one of theaforementioned (1) to (6), for example, with respect to the nucleotidesequence of SEQ ID NO:1, there may be exemplified a DNA sequence havingany one of:

i) mutation to convert GAA of base Nos. 2109-2111 into AAA or AAG;

ii) mutation to convert CGC of base Nos. 900-902 into CAT or CAC, andGAA of 903-905 into AAA or AAG, respectively;

iii) mutation to convert TCT of base Nos. 1098-1100 into TTT or TTC, GAAof 1101-1103 into AAA or AAG, ATG of 1887-1889 into ATT, ATC or ATA, andGAA of 2646-2648 into AAA or AAG, respectively;

iv) mutation to convert GCG of 2835-2837 into any one of ACT, ACC, ACAand ACG; and

v) mutation to convert CGT of 1548-1550 into TGT or TGC; and

vi) mutation to convert AAA of 2094-2096 into TCT, TCC, TCA or TCG.

Such a mutant gene is obtained such that a recombinant DNA, which isobtained by ligating a phosphoenolpyruvate carboxylase gene as a wildtype enzyme gene or having another mutation with a vector DNA adaptableto a host, is subjected to a mutation treatment, to perform screeningfrom transformants by the recombinant DNA. Alternatively, it is alsoacceptable that a microorganism which produces a wild type enzyme issubjected to a mutation treatment, a mutant strain which produces amutant enzyme is created, and then a mutant gene is screened from themutant strain. For the mutation treatment of the recombinant DNA,hydroxylamine and the like may be used. Further, when an microorganismitself is subjected to a mutation treatment, a drug or a method usuallyused for artificial mutation may be used.

Further, in accordance with methods such as the Overlapping Extensionmethod (Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease,L. R., Gene, 77, 51-59 (1989)), the site specific mutation method(Kramer, W. and Frits, H. J., Meth. in Enzymol., 154, 350 (1987);Kunkel, T. A. et al., Meth. in Enzymol., 154, 367 (1987)) and the like,the aforementioned mutant gene can be also obtained by introducingmutation such as amino acid replacement, insertion, deletion and thelike into a phosphoenolpyruvate carboxylase gene as a wild type enzymegene or having another mutation. These methods are based on a principlethat a non-mutated gene DNA is used as a template, and a synthetic DNAcontaining a mismatch at a mutation point is used as one of primers soas to synthesize complemental strands of the aforementioned gene DNA,thereby mutation is introduced. By using these methods, it is possibleto cause intended mutation at an aimed site.

Alternatively, a sequence, which has restriction enzyme cleavage ends atboth termini and includes both sides of a mutation point, issynthesized, and exchanged for a corresponding portion of a non-mutatedgene, thereby mutation can be introduced (cassette mutation method).

The phosphoenolpyruvate carboxylase gene, which is the wild type enzymegene or has another mutation to be used for introduction of mutation,may be any one provided that it is a gene coding for thephosphoenolpyruvate carboxylase of the microorganism belonging to thegenus Escherichia, which is preferably determined for its base sequenceand cloned. When it has not been cloned, a DNA fragment containing thegene can be amplified and isolated by using the PCR method and the like,followed by using a suitable vector to achieve cloning.

As the gene as described above, for example, there may be exemplified agene of Escherichia coli having been cloned and determined for its basesequence (Fujita, N., Miwa, T., Ishijima, S., Izui, K. and Katsuki, H.,J. Biochem., 95, 909-916 (1984)). The sequence in the coding region ofthis gene is as shown in SEQ ID NO:1 (base Nos. 237-2888).

Screening of a host harboring the mutant gene can be performed by usingan analog compound of aspartic acid. The analog compound preferably hasthe following properties. Namely, it exhibits a growth inhibitory actionagainst a microorganism belonging to the genus Escherichia whichproduces a wild type phosphoenolpyruvate carboxylase, the aforementionedgrowth inhibitory action is recovered by existence of L-glutamic acid orL-aspartic acid, and it inhibits wild type phosphoenolpyruvatecarboxylase activity.

If a mutant strain beeing resistant to the analog compound mentionedabove is selected from microorganism belonging to the genus Escherichia,for example, Escherichia coli HB101 producing wild typephosphoenolpyruvate carboxylase using inhibition of growth of themicroorganism as an index, it is much likely to obtain a hostmicroorganism which produces phosphoenolpyruvate carboxylase withdesensitized feedback inhibition by aspartic acid.

It is proposed, as a general structure of an inhibitor ofphosphoenolpyruvate carboxylase, that a C4 dicarboxylic acid structureis essentially provided. From such a viewpoint, various compounds weresubjected to screening by the present inventors. Escherichia coli HB101was cultivated in an LB medium, and transferred to M9 media (containing20 μg/ml of thiamine and 3 μg/ml of each of Leu and Pro) containing anyone of DL-2-amino-4-phosphonobutyric acid, bromosuccinic acid,meso-2,3-dibromosuccinic acid, 2,2-difluorosuccinic acid, 3-bromopyruvicacid, α-ketobutyric acid, α-ketoadipinic acid,DL-threo-β-hydroxyaspartic acids L-aspartic acid-β-metyl ester,α-metyl-DL-aspartic acid, 2,3-diaminosuccinic acid or asparticacid-β-hydrazide, and absorbance of the medium was measured at 660 nmwith the passage of time, thereby growth was monitored.

Further, when these compounds were present at their growth inhibitoryconcentrations, it was investigated whether or not the inhibition wasrecovered by addition of nucleic acids (each of uridine, adenosine: 10mg/dl), glutamic acid or amino acids of the aspartic acid family (Asp:0.025%, each of Met, Thr, Lys: 0.1%).

As a result, three compounds: 3-bromopyruvate (3BP) (1),aspartate-β-hydrazide (AHY) (2), and DL-threoβ-hydroxyaspartate (βHA)(3) were selected. ##STR1##

Growth inhibition of Escherichia coli by these analog compounds is shownin FIGS. 1-3. Further, growth recovery of Escherichia coli, in the caseof addition of the aforementioned inhibition recovering substances aloneor as a mixture of 2 species or 3 species, is shown in FIGS. 4-6. Inaddition, as a control, growth in the case of addition of the inhibitionrecovering substance in the absence of the inhibitory substance is shownin FIG. 7. Incidentally, in FIGS. 4-7, additives 1, 2 and 3 indicatenucleic acids, glutamic acid or amino acids of the aspartic acid family,respectively.

Further, inhibition of activity by the analog compound onphosphoenolpyruvate carboxylase was investigated. Crude enzyme wasprepared from an Escherichia coli HB101 strain in accordance with amethod described in The Journal of Biochemistry, Vol. 67, No. 4 (1970),and enzyme activity was measured in accordance with a method describedin Eur. J. Biochem., 202, 797-803 (1991).

Escherichia coli HB101 cultivated in an LB medium was disrupted, and asuspension was centrifuged to obtain a supernatant which was used as acrude enzyme solution. Measurement of enzyme activity was performed bymeasuring decrease in absorbance at 340 nm while allowingacetyl-coenzyme A known to affect the activity to exist at aconcentration of 0.1 mM in a measurement system containing 2 mMpotassium phosphoenolpyruvate, 0.1 mM NADH, 0.1M Tris-acetate (pH 8.5),1.5 U malate dehydrogenase and crude enzyme. Results are shown in FIG.8.

According to the results as above, it is apparent that theaforementioned three compounds inhibit growth of Escherichia coli, thisinhibition cannot be recovered by nucleic acids alone, but theinhibition can be recovered by addition of glutamic acid or amino acidsof the aspartic acid family. Therefore, these analog compounds werepostulated to be selective inhibitors of phosphoenolpyruvatecarboxylase. As shown in Examples described below, by using thesecompounds, the present invention has succeeded in selection of anEscherichia coli which produces the mutant phosphoenolpyruvatecarboxylase.

When a transformant having an aimed mutant enzyme gene is screened byusing the aforementioned compounds, and a recombinant DNA is recovered,then the mutant enzyme gene is obtained. Alternatively, in the case of amutation treatment of an microorganism itself, when a mutant strainhaving an aimed mutant enzyme gene is screened by using theaforementioned compounds, a DNA fragment containing the aimed mutantenzyme gene is isolated from the strain, and it is ligated with asuitable vector, then the mutant enzyme gene is obtained.

On the other hand, as a result of diligent investigation by the presentinventors taking notice of importance of an arginine residue in anaspartate binding protein of Escherichia coli (Krikos, A., Mouth, N.,Boyd, A. and Simon, M. I. Cell, 33, 615-622 (1983), Mowbray, S. L andKoshland, D. E. J. Biol. Chem., 264, 15638-15643 (1990), Milburn, M. V.,Prive, G. G., Milligan, D. L., Scott, W. G., Yeh, J., Jancarik, J.,Koshland, D. E. and Kim, S. H., Science, 254, 1342-1347 (1991)), it hasbeen found that inhibition by aspartic acid is substantiallydesensitized by converting 438th arginine of phosphoenolpyruvatecarboxylase into cysteine. In order to convert 438th arginine intocysteine, a codon of 438th arginine of a gene coding forphosphoenolpyruvate carboxylase may be converted into a codon ofcysteine. For example, in SEQ ID NO:1, CGT of nucleotide numbers of1548-1550 may be converted into TGT or TGC.

Further, the present inventors performed chemical modification of lysineresidues of phosphoenolpyruvate carboxylase by using2,4,6-trinitrobenzenesulfonic acid (TNBS) which is a compound tochemically modify lysine residues of a protein. During modificationreaction, malic acid capable of serving as an inhibitor ofphosphoenolpyruvate carboxylase was allowed to exist together. Namely,it was assumed that a lysine residue in the vicinity of a bindingposition of phosphoenolpyruvate carboxylase would be protected by boundmalic acid and not be subjected to chemical modification. As a result,it was suggested that a 620th lysine residue was important for malicacid to bind phosphoenolpyruvate carboxylase, and it was found that thefeedback inhibition by aspartic acid was desensitized while maintainingthe enzyme activity of phosphoenolpyruvate carboxylase by converting the620th lysine residue into a serine residue. In order to convert the620th lysine residue into the serine residue, a codon of 620th lysine ofthe gene coding for phosphoenolpyruvate carboxylase may be convertedinto a codon of serine. For example, in SEQ ID NO:1, AAA havingnucleotide numbers of 2094-2096 may be replaced with TCT, TCC, TCA, TCG,AGT or AGC.

In accordance with methods such as the Overlapping Extension method (Ho,S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R., Gene,77, 51-59 (1989)), the site specific mutation method (Kramer, W. andFrits, H. J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T. A. et al.,Meth. in Enzymol., 154, 367 (1987)) and the like, conversion of thecodon can be also achieved by introducing mutation such as amino acidreplacement, insertion, deletion and the like into a phosphoenolpyruvatecarboxylase gene as a wild type enzyme gene or having another mutation.These methods are based on a principle that a non-mutated gene DNA isused as a template, and a synthetic DNA containing a mismatch at amutation point is used as one of primers so as to synthesizecomplemental strands of the aforementioned gene DNA, thereby mutation isintroduced. By using these methods, it is possible to cause intendedmutation at an aimed site.

Alternatively, a sequence, which has restriction enzyme cleavage ends atboth termini and contains both sides of a mutation point, issynthesized, and exchanged for a corresponding portion of a non-mutatedgene, thereby mutation can be introduced (cassette mutation method).

The DNA fragment coding for the phosphoenolpyruvate carboxylase withmutation introduced as described above is expressed by using a suitablehost-vector system, thereby it is possible to produce a mutant enzyme.Alternatively, even by performing transformation by integrating the DNAfragment of the present invention into a host chromosomal DNA, an aimedmutant enzyme can be produced.

As the host, there may be exemplified microorganisms belonging to thegenus Escherichia, for example, Escherichia coli, coryneform bacteriaand the like. The coryneform bacteria include bacteria belonging to thegenus Corynebacterium, bacteria belonging to the genus Brevibacteriumhaving been hitherto classified into the genus Brevibacterium but beingunited as bacteria belonging to the genus Corynebacterium at present,and bacteria belonging to the genus Brevibacterium closely related tobacteria belonging to the genus Corynebacterium. Incidentally, hostswhich are preferable for amino acid production will be described below.

On the other hand, as the vector DNA, a plasmid vector is preferable,and those capable of self-replication in a host cell are preferable.When the host is Escherichia coli, for example, pUC19, pUC18, pBR322,pHSG299, pHSG399, RSF1010 and the like are exemplified. Alternatively, avector of phage DNA can be also utilized.

Further, when the host is the coryneform bacteria, vectors which can beused and hosts which harbor them are exemplified below. Incidentally,deposition numbers of international depositories are shown inparentheses.

pAJ655 Escherichia coli AJ11882 (FERM BP-136)

Corynebacterium glutamicum SR8201 (ATCC 39135)

pAJ1844 Escherichia coli AJ11883 (FERM BP-137)

Corynebacterium glutamicum SR8202 (ATCC 39136)

pAJ611 Escherichia coli AJ11884 (FERM BP-138)

pAJ3148 Corynebacterium glutamicum SR8203 (ATCC 39137)

pAJ440 Bacillus subtilis AJ11901 (FERM BP-140)

These vectors may be obtained from the deposited microorganisms asfollows. Cells collected at the logarithmic growth phase are subjectedto bacteriolysis by using lysozyme and SDS, and centrifuged at 30000×gto obtain a supernatant solution from a lysate, to which polyethyleneglycol is added to perform separation and purification of the vectors bymeans of cesium chloride-ethidium bromide equilibrium density gradientcentrifugation.

In order to transform Escherichia coli with a recombinant vectorobtained by inserting the DNA sequence of the present invention into theaforementioned vector, it is possible to use a method usually used fortransformation of Escherichia coli, such as a method in which cells aretreated with calcium chloride to enhance permeability of DNA (Mandel, M.and Higa, A., J. Mol. Biol., 53, 159 (1977)) and the like.

Further, as a method for transforming the coryneform bacteria, there isthe aforementioned method in which cells are treated with calciumchloride, or a method in which incorporation is performed at a specifiedgrowth period in which cells can incorporate DNA (report in relation toBacillus subtilis by Duncan, C. H. at al.). Further, incorporation intobacterial cells can be achieved by forming protoplasts or spheroplastsof DNA recipients which easily incorporate plasmid DNA. These are knownfor Bacillus subtilis, Actinomyces and yeast (Chang, S. et al., Molec.Gen. Genet., 168, 111 (1979), Bibb et al., Nature, 274, 398 (1978),Hinnen, A. et al., Proc. Natl. Acad. Sci. USA, 75 1929 (1978)).Additionally, a method for transforming coryneform bacteria is disclosedin Japanese Patent Laid-open No. 2-207791.

In order to express the DNA sequence of the present invention in theaforementioned hosts, a promoter such as lac, trp, PL and the like whichefficiently works in microorganisms may be used, or when the DNAsequence of the present invention contains a promoter of thephosphoenolpyruvate carboxylase gene, it may be used as it is.Alternatively, when the coryneform bacterium is used as the host, it isalso possible to use a known trp promoter originating from a bacteriumbelonging to the genus Brevibacterium (Japanese Patent Laid-open No.62-244382) and the like.

Further, as described above, it is acceptable that the DNA sequence ofthe present invention is inserted into the vector DNA capable ofself-replication and introduced into the host to allow the host toharbor it as a plasmid, and it is also acceptable that the DNA sequenceof the present invention is integrated into a chromosome of anmicroorganism by means of a method using transposon (Berg, D. E. andBerg, C. M., Bio/Technol., 1, 417 (1983)), Mu phage (Japanese PatentLaid-open No. 2-109985) or homologous recombination (Experiments inMolecular Genetics, Cold Spring Harbor Lab. (1972)). In addition, inorder to integrate the DNA of the present invention into the coryneformbacteria, it is possible to utilize a temperature-sensitive plasmiddisclosed in Japanese Patent Laid-open No. 5-7491.

When the microorganism transformed with the DNA sequence of the presentinvention as described above is cultivated, and this DNA sequence isexpressed, then a mutant enzyme is obtained. It becomes apparent, bymeasuring the activity by adding aspartic acid to an enzyme reactionsystem, whether or not the mutant enzyme thus obtained has desensitizedfeedback inhibition by aspartic acid. It is possible for the measurementof the enzyme activity to use a spectrometric method (Yoshinage, T.,Izui, K. and Katsuki, H., J. Biochem., 68, 747-750 (1970)) and the like.

Further, the DNA sequence of the present invention codes for the mutantenzyme in which feedback inhibition by aspartic acid is desensitized, sothat the microorganism harboring this DNA sequence can be utilized forefficient fermentative production of amino acids of the aspartic acidfamily and the glutamic acid family as described below.

Escherichia coli AJ12907, AJ12908, AJ12909 and AJ12910 harboring themutant enzyme genes obtained in Examples described below are depositedin National Institute of Bioscience and Human Technology of Agency ofIndustrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan; zip code 305) on Aug. 3, 1993 under the depositionnumbers of FERM P-13774, FERM P-13775, FERM P-13776 and FERM P-13777,transferred from the original deposition to international depositionbased on Budapest Treaty on Jul. 11, 1994 and has been deposited asdeposition numbers of FERM BP-4734, FERM BP-4735, FERM BP-4736, FERMBP-4737, respectively in this order.

<3> Production method of amino acids

Amino acids can be produced by cultivating the microorganism harboringthe DNA sequence of the present invention in a preferable medium, andseparating generated amino acids. As such amino acids, there may beexemplified L-lysine, L-threonine, L-methionine, L-isoleucine,L-glutamic acid, L-arginine and L-proline.

Preferable hosts into which the DNA sequence of the present invention isintroduced to be used for production of each of the amino acids, and acultivation method will be exemplified below.

(1) Hosts preferable for the amino acid production method of the presentinvention

(i) Hosts preferable for L-lysine production

As the host to be used for L-lysine production according to the presentinvention, there may be exemplified bacteria belonging to the genusEscherichia, preferably L-lysine-producing Escherichia coli. Concretely,a mutant strain having resistance to a lysine analog can be exemplified.Such a lysine analog is those which inhibit growth of microorganismsbelonging to the genus Escherichia, however, the suppression is totallyor partially desensitized provided that L-lysine co-exits in the medium.For example, there are oxalysine, lysine hydroxamate,S-(2-aminoethyl)-cysteine (hereinafter abbreviated as "AEC"),γ-methyllysine, α-chlorocaprolactam and the like. Mutant strains havingresistance to these lysine analogs can be obtained by applying anordinary artificial mutation treatment to microorganisms belonging tothe genus Escherichia. Concretely, as a bacterial strain to be used forL-lysine production, there may be exemplified Escherichia coli AJ11442(deposited as FERM P-5084, see lower-left column on page 471 in JapanesePatent Laid-open No. 56-18596).

On the other hand, various artificial mutant strains of coryneformbacteria which have been used as L-lysine-producing bacteria can be usedfor the present invention. Such artificial mutant strains are asfollows: AEC resistant mutant strain; mutant strain which requires aminoacid such as L-homoserine for its growth (Japanese Patent PublicationNos. 48-28078 and 56-6499); mutant strain which exhibits resistance toAEC and requires amino acid such as L-leucine, L-homoserine, L-proline,L-serine, L-arginine, L-alanine, L-valine and the like (U.S. Pat. Nos.3,708,395 and 3,825,472); L-lysine-producing mutant strain whichexhibits resistance to DL-α-amino-ε-caprolactam, α-amino-lauryllactam,quinoid and N-lauroylleucine; L-lysine-producing mutant strain whichexhibits resistance to an inhibitor of oxaloacetate decarboxylase orrespiratory system enzyme (Japanese Patent Laid-open Nos. 50-53588,50-31093, 52-102498, 53-86089, 55-9783, 55-9759, 56-32995 and 56-39778,and Japanese Patent Publication Nos. 53-43591 and 53-1833);L-lysine-producing mutant strain which requires inositol or acetic acid(Japanese Patent Laid-open Nos. 55-9784 and 56-8692); L-lysine-producingmutant strain which exhibits sensitivity to fluoropyruvate ortemperature not less than 34° C. (Japanese Patent Laid-open Nos. 55-9783and 53-86090); and mutant strain of Brevibacterium or Corynebacteriumwhich exhibits resistance to ethylene glycol and produces L-lysine (seeU.S. Pat. application Ser. No. 333,455).

Followings are exemplified as concrete coryneform bacteria to be usedfor lysine production:

Brevibacterium lactofermentum AJ12031 (FERM-BP277), see page 525 inJapanese Patent Laid-open No. 60-62994;

Brevibacterium lactofermentum ATCC 39134, see lower-right column on page473 in Japanese Patent Laid-open No. 60-62994;

Brevibacterium lactofermentum AJ3463 (FERM-P1987), see Japanese PatentPublication No. 51-34477.

In addition, wild strains of coryneform bacteria described below can bealso used for the present invention in the same manner.

Corynebacterium acetoacidophilum ATCC 13870

Corynebacterium acetoglutamicum ATCC 15806

Corynebacterium callunae ATCC 15991

Corynebacterium glutamicum ATCC 13032

ATCC 13060

(Brevibacterium divaricatum) ATCC 14020

(Brevibacterium lactofermentum) ATCC 13869

(Corynebacterium lilium) ATCC 15990

Corynebacterium melassecola ATCC 17965

Brevibacterium saccharolyticum ATCC 14066

Brevibacterium immariophilum ATCC 14068

Brevibacterium roseum ATCC 13825

Brevibacterium flavum ATCC 13826

Brevibacterium thiogenitalis ATCC 19240

Microbacterium ammoniaphilum ATCC 15354

(ii) Hosts preferable for L-threonine production

Escherichia coli B-3996 (RIA 1867), see Japanese Patent Laid-open No.3-501682 (PCT);

Escherichia coli AJ12349 (FERM-P9574), see upper-left column on page 887in Japanese Patent Laid-open No. 2-458;

Escherichia coli AJ12351 (FERM-P9576), see lower-right column on page887 in Japanese Patent Laid-open No. 2-458;

Escherichia coli AJ12352 (FERM P-9577), see upper-left column on page888 in Japanese Patent Laid-open No. 2-458;

Escherichia coli AJ11332 (FERM P-4898), see upper-left column on page889 in Japanese Patent Laid-open No. 2-458;

Escherichia coli AJ12350 (FERM P-9575), see upper-left column on page889 in Japanese Patent Laid-open No. 2-458;

Escherichia coli AJ12353 (FERM P-9578), see upper-right column on page889 in Japanese Patent Laid-open No. 2-458;

Escherichia coli AJ12358 (FERM P-9764), see upper-left column on page890 in Japanese Patent Laid-open No. 2-458;

Escherichia coli AJ12359 (FERM P-9765), see upper-left column on page890 in Japanese Patent Laid-open No. 2-458;

Escherichia coli AJ11334 (FERM P-4900), see column 6 on page 201 inJapanese Patent Publication No. 1-29559;

Escherichia coli AJ11333 (FERM P-4899), see column 6 on page 201 inJapanese Patent Publication No. 1-29559;

Escherichia coli AJ11335 (FERM P-4901), see column 7 on page 202 inJapanese Patent Publication No. 1-29559.

Following bacterial strains are exemplified as the coryneform bacteria:

Brevibacterium lactofermentum AJ11188 (FERM P-4190), see upper-rightcolumn on page 473 in Japanese Patent Laid-open No. 60-87788;

Corynebacterium glutamicum AJ11682 (FERM BP-118), see column 8 on page230 in Japanese Patent Publication No. 2-31956;

Brevibacterium flavum AJ11683 (FERM BP-119), see column 10 on page 231in Japanese Patent Publication No. 2-31956.

(iii) Hosts preferable for L-methionine production

Following bacterial strains are exemplified for L-methionine production:

Escherichia coli AJ11457 (FERM P-5175), see upper-right column on page552 in Japanese Patent Laid-open No. 56-35992;

Escherichia coli AJ11458 (FERM P-5176), see upper-right column on page552 in Japanese Patent Laid-open No. 56-35992;

Escherichia coli AJ11459 (FERM P-5177), see upper-right column on page552 in Japanese Patent Laid-open No. 56-35992;

Escherichia coli AJ11539 (FERM P-5479), see lower-left column on page435 in Japanese Patent Laid-open No. 56-144092;

Escherichia coli AJ11540 (FERM P-5480), see lower-left column on page435 in Japanese Patent Laid-open No. 56-144092;

Escherichia coli AJ11541 (FERM P-5481), see lower-left column on page435 in Japanese Patent Laid-open No. 56-144092;

Escherichia coli AJ11542 (FERM P-5482), see lower-left column on page435 in Japanese Patent Laid-open No. 56-144092.

(iv) Hosts preferable for L-aspartic acid production

Following bacterial strains are exemplified for L-aspartic acidproduction:

Brevibacterium flavum AJ3859 (FERM P-2799), see upper-left column onpage 524 in Japanese Patent Laid-open No. 51-61689;

Brevibacterium lactofermentum AJ3860 (FERM P-2800), see upper-leftcolumn on page 524 in Japanese Patent Laid-open No. 51-61689;

Corynebacterium acetoacidophilum AJ3877 (FERM-P2803), see upper-leftcolumn on page 524 in Japanese Patent Laid-open No. 51-61689;

Corynebacterium glutamicum AJ3876 (FERM P-2802), see upper-left columnon page 524 in Japanese Patent Laid-open No. 51-61689.

(v) Hosts preferable for L-isoleucine production

Escherichia coli KX141 (VKPM-B4781) (see 45th paragraph in JapanesePatent Laid-open No. 4-33027) is exemplified as the bacteria belongingto the genus Escherichia, and Brevibacterium lactofermentum AJ12404(FERM P-10141) (see lower-left column on page 603 in Japanese PatentLaid-open No. 2-42988) and Brevibacterium flavum AJ12405 (FERM P-10142)(see lower-left column on page 524 in Japanese Patent Laid-open No.2-42988) are exemplified as the coryneform bacteria.

(vi) Hosts preferable for L-glutamic acid production

Following bacterial strains are exemplified as the bacteria belonging tothe genus Escherichia:

Escherichia coli AJ12628 (FERM P-12380), see French Patent PublicationNo. 2 680 178 (1993);

Escherichia coli AJ12624 (FERM P-12379), see French Patent PublicationNo. 2 680 178 (1993).

Following bacterial strains are exemplified as the coryneform bacteria:

Brevibacterium lactofermentum AJ12745 (FERM BP-2922), see lower-rightcolumn on page 561 in Japanese Patent Laid-open No. 3-49690;

Brevibacterium lactofermentum AJ12746 (FERM BP-2923), see upper-leftcolumn on page 562 in Japanese Patent Laid-open No. 3-49690;

Brevibacterium lactofermentum AJ12747 (FERM BP-2924), see upper-leftcolumn on page 562 in Japanese Patent Laid-open No. 3-49690;

Brevibacterium lactofermentum AJ12748 (FERM BP-2925), see upper-leftcolumn on page 562 in Japanese Patent Laid-open No. 3-49690;

Brevibacterium flavum ATCC 14067, see Table 1 on page 3 in JapanesePatent Laid-open No. 5-3793;

Corynebacterium glutamicum ATCC 21492, see Table 1 on page 3 in JapanesePatent Laid-open No. 5-3793.

(vii) Hosts preferable for L-arginine production

Following bacterial strains are exemplified as the bacteria belonging tothe genus Escherichia:

Escherichia coli AJ11593 (FERM P-5616), see upper-left column on page468 in Japanese Patent Laid-open No. 57-5693;

Escherichia coli AJ11594 (FERM P-5617), see upper-right column on page468 in Japanese Patent Laid-open No. 57-5693.

Following bacterial strains are exemplified as the coryneform bacteria:

Brevibacterium flavum AJ12144 (FERM P-7642), see column 4 on page 174 inJapanese Patent Publication No. 5-27388;

Corynebacterium glutamicum AJ12145 (FERM P-7643), see column 4 on page174 in Japanese Patent Publication No. 5-27388;

Brevibacterium flavum ATCC 21493, see Table 1 on page 3 in JapanesePatent Laid-open No. 5-3793;

Corynebacterium glutamicum ATCC 21659, see Table 1 on page 3 in JapanesePatent Laid-open No. 5-3793.

(viii) Hosts preferable for L-proline production

Following bacterial strains are exemplified as the bacteria belonging tothe genus Escherichia:

Escherichia coli AJ11543 (FERM P-5483), see lower-left column on page435 in Japanese Patent Laid-open No. 56-144093;

Escherichia coli AJ11544 (FERM P-5484), see lower-left column on page435 in Japanese Patent Laid-open No. 56-144093.

Following bacterial strains are exemplified as the coryneform bacteria:

Brevibacterium lactofermentum AJ11225 (FERM P-4370), see upper-leftcolumn on page 473 in Japanese Patent Laid-open No. 60-87788;

Brevibacterium flavum AJ11512 (FERM P-5332), see column 2 on page 185 inJapanese Patent Publication No. 62-36679;

Brevibacterium flavum AJ11513 (FERM P-5333), see column 2 on page 185 inJapanese Patent Publication No. 62-36679;

Brevibacterium flavum AJ11514 (FERM P-5334), see column 2 on page 185 inJapanese Patent Publication No. 62-36679;

Corynebacterium glutamicum AJ11522 (FERM P-5342), see column 2 on page185 in Japanese Patent Publication No. 62-36679;

Corynebacterium glutamicum AJ11523 (FERM P-5343), see column 2 on page185 in Japanese Patent Publication No. 62-36679.

(2) Cultivation method

The method for cultivating the aforementioned hosts is not especiallydifferent from a cultivation method for amino acid-producingmicroorganisms in the prior art. Namely, an ordinary medium is usedcontaining a carbon source, a nitrogen source and inorganic ions, andoptionally organic trace nutrients such as amino acids, vitamins and thelike.

As the carbon source, glucose, sucrose, lactose and the like, as well asstarch hydrolysate, whey, molasses and the like containing them may beused. As the nitrogen source, ammonia gas, aqueous ammonium, ammoniumsalt and the like can be used. Incidentally, when a nutrient requiringmutant strain for amino acids or the like is used as the host, it isnecessary to suitably add the nutrient such as amino acid or the likerequired by the strain to the medium. An example of the medium forlysine production is shown in Table 1 below as a medium to be used foramino acid production. Incidentally, calcium carbonate is added to othercomponents after being separately sterilized.

                  TABLE 1    ______________________________________    Medium component Blending amount    ______________________________________    glucose          5           g/dl    (NH.sub.4).sub.2 SO.sub.4                     2.5         g/dl    KH.sub.2 PO.sub.4                     0.2         g/dl    MgSO.sub.4.7H.sub.2 O                     0.1         g/dl    yeast extract    0.05        g/dl    thiamine hydrochloride                     1           μg/l    biotin           300         μg/l    FeSO.sub.4.7H.sub.2 O                     1           mg/dl    MnSO.sub.4.4H.sub.2 O                     1           mg/dl    calcium carbonate                     2.5         g/dl    (pH 7.0)    ______________________________________

The cultivation is performed until generation and accumulation of aminoacids substantially stop while suitably controlling pH and temperatureof the medium under an aerobic condition. In order to collect aminoacids thus accumulated in the cultivated medium, an ordinary method canbe applied.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows growth inhibition by 3-bromopyruvate.

FIG. 2 shows growth inhibition by aspartate-β-hydrazide.

FIG. 3 shows growth inhibition by DL-threo-β-hydroxyaspartate.

FIG. 4 shows effects of inhibition recovering substances on3-bromopyruvate.

FIG. 5 shows effects of inhibition recovering substances onaspartate-β-hydrazide.

FIG. 6 shows effects of inhibition recovering substances onDL-threo-β-hydroxyaspartate.

FIG. 7 shows influences exerted on growth by growth recovering factors.

FIG. 8 shows inhibition of phosphoenolpyruvate carboxylase by growthinhibitory substances.

FIG. 9 shows inhibition of phosphoenolpyruvate carboxylase of thepresent invention by aspartic acid.

FIG. 10 shows inhibition of phosphoenolpyruvate carboxylase of thepresent invention by aspartic acid.

FIG. 11 shows a method for introducing mutation into aphosphoenolpyruvate carboxylase gene.

FIG. 12 shows influences exerted by aspartic acid on acitivities of wildtype and mutant phosphoenolpyruvate carboxylase in which 438th argininewas substituted with cysteine counted from the N-terminus.

FIG. 13 shows the influence exerted by (a) 1-10 mM and (b) 1-50 mM ofaspartic acid on the activities of the wild-type (solid circles) of E.coli phosphoenolpyruvate carboxylase and its mutants Lys-620Ser(diamonds), Lys-650Ala (open circles), and Lys-491Ala (squares).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained more concretely below withreference to Examples.

EXAMPLE 1 Acquisition of Mutant Phosphoenolpyruvate Carboxylase Gene

A mutant gene was prepared by using a plasmid pS2 obtained by insertinga phosphoenolpyruvate carboxylase gene having been cloned and determinedfor its base sequence into a SalI site of a vector plasmid pBR322. pS2has an ampicillin resistance gene as a drug resistance marker gene(Sabe, H. et al., Gene, 31, 279-283 (1984)). The nucleotide sequence ofthe phosphoenolpyruvate carboxylase gene contained in pS2 is the same asthat contained in the aforementioned plasmid pT2.

pS2 DNA was treated at 75° C. for 2 hours with a hydroxylamine treatingsolution (20 μg/ml pS2 DNA, 0.05M sodium phosphate (pH 6.0), 1 mM EDTA,0.4M hydroxylamine). Because of influence by pH on the hydroxylaminetreatment, 80 μl of 1M hydroxylamine.HCl and 1 mM EDTA solution having apH adjusted to 6.0 with sodium hydroxide, 100 μl of 0.1M sodiumphosphate (pH 6.0) and 1 mM EDTA solution, and TE (10 mM Tris-HCl, 1 mMEDTA) buffer containing 2 μg of pS2 DNA were mixed, to finally provide200 μl with water.

The aforementioned condition is a condition in which transformants has asurvival ratio of 0.2% based on a state before the treatment in anampicillin-containing medium when Escherichia coli HB101 is transformedwith pS2 after the treatment.

Escherichia coli HB101 was transformed with pS2 treated withhydroxylamine, which was spread on a solid plate medium containingampicillin to obtain about 10000 colonies of transformants. They weresuspended in a liquid medium, and spread on a solid plate mediumcontaining any one of 3-bromopyruvate (3BP), aspartate-β-hydroxamate(AHX), aspartate-β-hydrazide (AHY) and DL-threo-β-hydroxyaspartate (βHA)as the analog compounds of aspartic acid at a concentration near aminimal inhibitory concentration to give 10³ to 10⁵ cells per one mediumplate, and growing colonies were selected.

From 100 strains of analog compound resistant strains thus obtained,phosphoenolpyruvate carboxylase produced by each of them was partiallypurified in accordance with a method described in The Journal ofBiochemistry, Vol. 67, No. 4 (1970), and inhibition of enzyme activityby the analog compounds was investigated. Measurement of the enzymeactivity was performed in the same manner as described above.

Further, plasmids were isolated from bacterial strains producing mutantenzymes with activities not inhibited by the analog compounds, and wereintroduced into Escherichia coli PCR1 as a phosphoenolpyruvatecarboxylase deficient strain (Sabe, H. et al., Gene, 31, 279-283(1984)), to confirm production of the mutant enzymes.

Five transformants harboring mutant enzyme genes were thus obtained. Asa result of determination of base sequences of these genes, 2 strainshad the same mutation, and 4 kinds of mutant genes were obtained. Thetransformants harboring them were designated as AJ12907, AJ12908,AJ12909 and AJ12910, and were deposited in National Institute ofBioscience and Human Technology of Agency of Industrial Science andTechnology1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan; zipcode 305) on Aug. 3, 1993 under the deposition numbers of FERM P-13774,FERM P-13775, FERM P-13776 and FERM P-13777, transferred from theoriginal deposition to international deposition based on Budapest Treatyon Jul. 11, 1994 and has been deposited as deposition numbers of FERMBP-4734, FERM BP-4735, FERM BP-4736, FERM BP-4737, respectively in thisorder. Further, the plasmids possessed by them were designated as pBP5,pHA19, pBP122 and pR6 respectively in this order. Mutations possessed bythe phosphoenolpyruvate carboxylase genes contained in each of theplasmids are shown in Table 2. Numerical values in the table indicatenucleotide numbers or amino acid numbers in SEQ ID NO:1.

                  TABLE 2    ______________________________________                                  Amino acid replacement    Transformant             Plasmid   Mutation   associated with mutation    ______________________________________    AJ12907  pBP5      .sup.2109 G→A                                  .sup.625 Glu→Lys    AJ12908  PHA19     .sup.901 G→A                                  .sup.222 Arg→His                       .sup.903 G→A                                  .sup.223 Glu→Lys    AJ12909  pBP122    .sup.1099 C→T                                  .sup.288 Ser→Phe                       .sup.1101 G→A                                  .sup.289 Glu→Lys                       .sup.1889 G→A                                  .sup.551 Met→Ile                       .sup.2646 G→A                                  .sup.804 Glu→Lys    AJ12910  pR6       .sup.2835 G→A                                  .sup.867 Ala→Thr    ______________________________________

Incidentally, selection was performed for AJ12907 and AJ12909 in amedium containing 500 μg/ml of 3BP, for AJ12908 in a medium containing1000 μg/ml of βHA, and for AJ12910 in a medium containing 500 μg/ml ofAHY.

EXAMPLE 2 Mutant Phosphoenolpyruvate Carboxylase

Sensitivity to aspartic acid was investigated for phosphoenolpyruvatecarboxylases produced by the aforementioned 4 transformants. Thesebacterial strains are deficient in the phosphoenolpyruvate carboxylasegene originating from the host, so that produced phosphoenolpyruvatecarboxylase originates from the plasmid.

Sensitivity to aspartic acid was investigated in accordance with a knownmethod (Yoshinaga, T., Izui, K. and Katsuki, H., J. Biochem., 68,747-750 (1970)). Namely, as a result of measurement of the enzymeactivity produced by each of the transformants or Escherichia coliharboring pS2 in the presence of acetyl-coenzyme A known to affect theactivity in an activity measurement system at a concentration of 0.1 mMor 1 mM, sensitivity to aspartic acid was measured as shown in FIGS. 9and 10.

According to the result, it is apparent that the wild type enzyme losesits activity when aspartic acid is at a high concentration, while themutant phosphoenolpyruvate carboxylase of the present inventionsubstantially continues to maintain its activity.

EXAMPLE 3 Fermentative Production of L-threonine by Escherichia coliwith Introduced Mutant Phosphoenolpyruvate Carboxylase

As threonine-producing bacteria of Escherichia coli, B-3996 strain(Japanese Patent Laid-open No. 3-501682 (PCT)) has the highestproduction ability among those known at present. Thus upon evaluation ofthe mutant phosphoenolpyruvate carboxylase, B-3996 was used as the host.This B-3996 strain has been deposited in Research Institute for Geneticsand Industrial Microorganism Breeding under a registration number of RIA1867. Further, pBP5 was selected as the mutant phosphoenolpyruvatecarboxylase to be evaluated, which was subjected to an experiment.

The plasmid pBP5 having the mutant phosphoenolpyruvate carboxylase wasintroduced into Escherichia coli B-3996 in accordance with a method ofHanahan (J. Mol. Biol., Vol. 106, p577 (1983)), and a transformant wasisolated. As a control, Escherichia coli B-3996 was transformed in thesame manner with pS2 as the plasmid to express the wild typephosphoenolpyruvate carboxylase gene.

When Escherichia coli B-3996 and the transformants therefrom wererespectively inoculated in a 500 ml of Sakaguchi flask poured with 20 mlof a medium having a composition in Table 3, and cultivated at 37° C.for 40 hours to investigate a production amount of L-threonine, thenresults shown in Table 4 were obtained. Incidentally, the aforementionedmedium was separated into two: glucose and MgSO₄.7H₂ O, and the othercomponents, and adjusted to have a pH of 7.0 with KOH followed byautoclaving at 115° C. for 10 minutes, and then, after mixing them,separately sterilized CaCO₃ was added by 30 g/l.

                  TABLE 3    ______________________________________    Component      Blending amount (g/l)    ______________________________________    glucose        40    (NH.sub.4).sub.2 SO.sub.4                   16    KH.sub.2 PO.sub.4                   1    MgSO.sub.4.7H.sub.2 O                   1    FeSO.sub.4.7H.sub.2 O                   0.01    MnSO.sub.4.5H.sub.2 O                   0.01    yeast extract (Difco)                   2    L-Met          0.5    CaCO.sub.3     30    ______________________________________

                  TABLE 4    ______________________________________                     Threonine production amount    Bacterial strain (g/l)    ______________________________________    Escherichia coli B-3996                     15.7    Escherichia coli B-3996/pS2                     15.8    Escherichia coli B-3996/pBP5                     16.8    ______________________________________

As clarified from the result, Escherichia coli B-3996/pBP5 harboring themutant enzyme expression plasmid having the DNA sequence of the presentinvention had an improved threonine-producing ability as compared withEscherichia coli B-3996/pS2 harboring the plasmid to express the wildtype enzyme.

EXAMPLE 4 Fermentative Production of L-glutamic Acid by Escherichia coliwith Introduced Mutant Phosphoenolpyruvate Carboxylase

As glutamic acid-producing bacteria of Escherichia coli, Escherichiacoli AJ-12628 described in Japanese Patent Laid-open No. 4-11461 has thehighest production ability among those known at present. Thus uponevaluation of the mutant phosphoenolpyruvate carboxylase, AJ-12628 wasused as the host.

The AJ-12628 strain has been deposited in National Institute ofBioscience and Human Technology of Agency of Industrial Science andTechnology under a registration number of FERM BP-385 Further, pBP5 wasselected as the mutant phosphoenolpyruvate carboxylase to be evaluated,which was subjected to an experiment.

The plasmid pBP5 having the mutant phosphoenolpyruvate carboxylase wasintroduced into Escherichia coli AJ-12628 in accordance with a method ofHanahan (J. Mol. Biol., Vol. 106, p577 (1983)), and a transformant wasisolated. In the same manner, a transformant of Escherichia coliAJ-12628 with pS2 was isolated.

When Escherichia coli AJ-12628 and the transformants therefrom wererespectively inoculated in a 500 ml of Sakaguchi flask poured with 20 mlof a medium having a composition in Table 5, and cultivated at 37° C.for 36 hours to investigate a production amount of L-glutamic acid, thenresults shown in Table 6 were obtained. Incidentally, the aforementionedmedium was separated into two: glucose and MgSO₄.7H₂ O, and the othercomponents, and adjusted to have a pH of 7.0 with KOH followed byautoclaving at 115° C. for 10 minutes, and then, after mixing them,separately sterilized CaCO₃ was added by 30 g/l.

                  TABLE 5    ______________________________________    Component      Blending amount (g/l)    ______________________________________    glucose        40    (NH.sub.4).sub.2 SO.sub.4                   16    KH.sub.2 PO.sub.4                   1    MgSO.sub.4.7H.sub.2 O                   1    FeSO.sub.4.7H.sub.2 O                   0.01    MnSO.sub.4.5H.sub.2 O                   0.01    yeast extract (Difco)                   2    CaCO.sub.3     30    ______________________________________

                  TABLE 6    ______________________________________                      Glutamic acid production    Bacterial strain  amount (g/l)    ______________________________________    Escherichia coli AJ-12628                      18.0    Escherichia coli AJ-12628/pS2                      18.3    Escherichia coli AJ-12628/pBP5                      19.6    ______________________________________

As clarified from the result, Escherichia coli AJ-12628/pBP5 harboringthe mutant enzyme expression plasmid having the DNA sequence of thepresent invention had an improved glutamate-producing ability ascompared with Escherichia coli AJ-12628/pS2 harboring the plasmid toexpress the wild type enzyme.

EXAMPLE 5 Production of L-lysine by Coryneform Bacterium with IntroducedMutant Phosphoenolpyruvate Carboxylase

In order to introduce and express the mutant gene in a coryneformbacterium, a promoter originating from a bacterium belonging to thegenus Brevibacterium was obtained, and was ligated with the mutant geneto prepare an expression type plasmid. Further, it was introduced into abacterium belonging to the genus Brevibacterium to perform production ofL-lysine.

<1> Acquisition of aspartokinase (AK) gene originating from bacteriumbelonging to the genus Brevibacterium

Chromosomal DNA was prepared according to an ordinary method from aBrevibacterium lactofermentum (Corynebacterium glutamicum) wild strain(ATCC 13869). An AK gene was amplified from the chromosomal DNA by PCR(polymerase chain reaction; see White, T. J. et al., Trends Genet., 5,185 (1989)). For DNA primers used in the amplification, anoligonucleotide of 23 mer (SEQ ID NO:3) and an oligonucleotide of 21 mer(SEQ ID NO:4) were synthesized to amplify a region of about 1643 bpcoding for the AK gene based on a sequence known in Corynebacteriumglutamicum (see Molecular Microbiology (1991) 5 (5), 1197-1204, Mol.Gen. Genet. (1990) 224, 317-324).

The synthesis of DNA was performed in accordance with an ordinaryphosphoamidite method (see Tetrahedron Letters (1981), 22, 1859) using aDNA synthesizer model 380B produced by Applied Biosystems Co. In the PCRreaction, DNA Thermal Cycler PJ2000 type produced by Takara Shuzo Co.,Ltd. was used, and gene amplification was performed by using Taq DNApolymerase in accordance with a method designated by the manufacturer.

An amplified gene fragment of 1643 kb was confirmed by agarose gelelectrophoresis, and then the fragment cut out from the gel was purifiedby an ordinary method, and was cleaved with restriction enzymes NruI(produced by Takara Shuzo Co., Ltd.) and EcoRI (produced by Takara ShuzoCo., Ltd.). pHSG399 (see Takeshita, S. et al.; Gene (1987), 61, 63-74)was used for a cloning vector for the gene fragment. pHSG399 was cleavedwith a restriction enzyme SmaI (produced by Takara Shuzo Co., Ltd.) anda restriction enzyme EcoRI, and ligated with the amplified AK genefragment.

Ligation of DNA was performed by a designated method by using a DNAligation kit (produced by Takara Shuzo Co., Ltd.). In such a manner, aplasmid was manufactured in which pHSG399 was ligated with the AK genefragment amplified from Brevibacterium chromosome. The plasmid havingthe AK gene originating from ATCC 13869 as the wild strain wasdesignated as p399AKY.

<2> Determination of base sequence of AK gene of Brevibacteriumlactofermentum

The AK plasmid, p399AKY was prepared, and the base sequence of the AKgene was determined. Determination of the base sequence was performed inaccordance with the method of Sanger et al. (F. Sanger et al.: Proc.Natl. Acad. Sci. USA, 74, 5463 (1977) and so forth). Results are shownin SEQ ID NO:5 and SEQ ID NO:7. The DNA fragments have two open readingframes which correspond to α-subunit and β-subunit of AK, respectively.In SEQ ID NO:5 and SEQ ID NO:7, amino acid sequences corresponding toeach of the open reading frames are shown together with nucleotidesequences. Further, only the amino acid sequences corresponding to eachof the open reading frames are shown in SEQ ID NO:6 and SEQ ID NO:8.

<3> Preparation of phosphoenolpyruvate carboxylase expression plasmid

SalI fragments of about 4.4 kb containing phosphoenolpyruvatecarboxylase genes were extracted from pS2 as the plasmid having the wildtype phosphoenolpyruvate carboxylase gene and pBP5 as the plasmid havingthe obtained mutant phosphoenolpyruvate carboxylase gene, and insertedinto a SalI site of a plasmid vector pHSG399 universally used forEscherichia coli. Manufactured plasmids were designated as pHS2 for thewild type and as pHBP5 for the mutant.

In order to convert pHS2 and pHPB5 into plasmids to express inBrevibacterium, a promoter and a replication origin of a plasmid forfunctioning in Brevibacterium were introduced. As the promoter, a genefragment containing one from 1st NruI site to 207th ApaLI site of thebase sequence, which was postulated to be a promoter region of thecloned AK gene, was extracted from p399AKY, and inserted into an AvaIsite located about 60 bp before the structural genes of pHS2 and pHBP5to allow the transcription direction to be in a regular direction.

Further, a gene fragment to enable autonomously replication of theplasmid in Brevibacterium, namely the replication origin of the plasmidwas introduced into a site located on the vector. A gene fragmentcontaining the replication origin of the plasmid was extracted from avector pHC4 for Brevibacterium (see paragraph No. 10 in Japanese PatentLaid-open No. 5-7491; Escherichia coli AJ12039 harboring the sameplasmid is deposited in National Institute of Bioscience and HumanTechnology of Agency of Industrial Science and Technology, to which adeposition number of FERM P12215 is given), and restriction enzyme sitesat both termini were modified into PstI sites by introduction oflinkers.

This fragment was introduced into a PstI site in a vector portion of theplasmid added with the promoter derived from Brevibacterium. Constructedphosphoenolpyruvate carboxylase-expressing plasmids were designated aspHS2B for a wild type phosphoenolpyruvate carboxylase plasmidoriginating from pS2 and as pHBP5B for a mutant phosphoenolpyruvatecarboxylase plasmid originating from pBP5, respectively.

<4> Production of L-lysine by using phosphoenolpyruvate carboxylaseexpression type plasmid

Prepared pHS2B and pHBP5B were respectively introduced into AJ3463 as anL-lysine-producing bacterium of Brevibacterium lactofermentum (seeJapanese Patent Publication No. 51-34477). For introduction of the gene,a transformation method employing electric pulse was used (see JapanesePatent Laid-open No. 2-207791). The host strain and transformants werecultivated with shaking for 72 hours at 31.5° C. in a lysine productionmedium having a composition in Table 7. The aforementioned medium wasprepared such that those except for CaCO₃ among the components listed inthe table were added to 1 l of water, and adjusted to have a pH of 8.0with KOH followed by autoclaving at 115° C. for 15 minutes, and thenCaCO₃ having been subjected to heat sterilization was further added.Accumulated amounts of L-lysine in the medium after cultivation areshown in Table 8.

                  TABLE 7    ______________________________________    Component       Blending amount in 1 L    ______________________________________    glucose         100          g    (NH.sub.4).sub.2 SO.sub.4                    55           g    soybean concentrate*                    35           m/l    KH.sub.2 PO.sub.4                    1            g    MgSO.sub.4.7H.sub.2 O                    1            g    Vitamin B1      20           g    biotin          5            g    nicotinic acid amide                    5            mg    FeSO.sub.4.7H.sub.2 O                    0.01         g    MnSO.sub.4.5H.sub.2 O                    0.01         g    CaCO.sub.3      50           g    ______________________________________     *product of Ajinomoto Co., Ltd. (trade name: Mamenou)

                  TABLE 8    ______________________________________                           Lysine production    Bacterial strain       amount (g/l)    ______________________________________    Brevibacterium lactofermentum AJ3463                           20.0    Brevibacterium lactofermentum AJ3463/pHS2B                           22.0    Brevibacterium lactofermentum AJ3463/pHBP5B                           25.0    ______________________________________

As shown in the result, Brevibacterium lactofermentum AJ3463/pHBP5Bharboring the mutant enzyme expression plasmid having the DNA sequenceof the present invention had an improved lysine-producing ability ascompared with Brevibacterium lactofermentum AJ3463/pHS2B harboring theplasmid to express the wild type enzyme.

EXAMPLE 6 Another Example of Mutant Phosphoenolpyruvate Carboxylase ofthe Present Invention and its Gene

<1> Preparation of mutant phosphoenolpyruvate carboxylase gene

Upon preparation of DNA coding for a mutant phosphoenolpyruvatecarboxylase, a phosphoenolpyruvate carboxylase gene cloned in a plasmidpT2 was used as a material.

A host, which is allowed to harbor the plasmid pT2, is preferablydeficient in phosphoenolpyruvate carboxylase gene in order to detectonly the activity of phosphoenolpyruvate carboxylase originating fromthe plasmid. Escherichia coli F15 (Hfr, recA1, met, Δ(ppc-argECBH),Tn10) was used as such a deficient strain. Escherichia coli AJ-12873,which is allowed to harbor pT2 in F15 strain, is deposited as FERMP-13752 in National Institute of Bioscience and Human Technology ofAgency of Industrial Science and Technology (1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken, Japan; zip code 305) on Jul. 15, 1993,transferred from the original deposition to international depositionbased on Budapest Treaty on Jul. 11, 1994 and has been deposited asdeposition number of FERM BP-4732. In addition, an entire base sequenceof pT2 is shown in SEQUENCE ID NO:1.

In order to replace a codon of 438th arginine of the phosphoenolpyruvatecarboxylase into a codon of cysteine by using pT2, the OverlappingExtension method (Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K.and Pease, L. R., Gene, 77, 51-59 (1989)) utilizing the PCR (PolymeraseChain Reaction) method was used.

Incidentally, the PCR method is a method in which an amplification cyclecomprising thermal denaturation of double strand DNA into single strandDNA, annealing of oligonucleotide primers corresponding to sequences atboth ends of a site aimed to be amplified and the aforementionedthermally denatured DNA, and polymerase reaction using theaforementioned oligonucleotides as primers is repeated, thereby theaforementioned DNA sequence is amplified in a manner of an exponentialfunction.

A region subjected to site specific mutation by the PCR method is shownin FIG. 11. The primers used in the present invention were 4 species ofa primer c (SEQUENCE ID NO:11, corresponding to base Nos. 1535-1554 inSEQUENCE ID NO:1) having a sequence in the vicinity of the codon of438th arginine, a primer b (SEQUENCE ID NO:10) having a sequencecomplement to the primer c, a primer a (SEQUENCE ID NO:9, correspondingto base Nos. 1185-1200 in SEQUENCE ID NO:1) having a sequence upstreamtherefrom, and a primer d (SEQUENCE ID NO:12, corresponding to base Nos.2327-2342 in SEQUENCE ID NO:1) having a sequence complement to adownstream sequence.

In the primer b and the primer c, the codon (CGT) of 438th arginine wasreplaced with a codon (TGT) of cysteine. This replacement may use TGCwhich is another codon of cysteine. Further, C of the third letter of acodon (AAC) of 435th asparagine was replaced with T, and hence an EcoRIsite was internally introduced with no replacement of amino acid, sothat a mutant plasmid could be selected by using it as an index.However, this mutation is not essential to the present invention.

When the PCR reaction was performed by using pT2 DNA as a template andthe primer a and the primer b as the primers, a fragment from theupstream of the mutation site to the mutation site (AB fragment in FIG.11) was amplified. Further, when the PCR reaction was performed by usingthe primer c and the primer d, a fragment downstream from the mutationsite (CD fragment in FIG. 11) was amplified. When each of the amplifiedproducts (AB, CD) was annealed again after thermal denaturation toperform a polymerase reaction, they were ligated to obtain a fragment(AD fragment in FIG. 11). Incidentally, the PCR reaction was performedby repeating 30 cycles of each comprising heating at 94° C. for 1 minutefollowed by denaturation (94° C., 1.5 minutes), annealing (50° C., 2minutes), and elongation reaction by polymerase (72° C., 3.5 minutes).In addition, reaction compositions are shown in Table 9.

                  TABLE 9    ______________________________________    Composition     PCR fragment    ((  ): final conc.)                    AB        CD      AD    ______________________________________    H.sub.2 O         53.5      53.5    53.5    10-fold reaction buffer                    10        10      10    mixture of 1.25 mM dNTP                    16        16      16    20 μM primer a (1 μM)                    5         --      5    20 μM primer b (1 μM)                    5         --      --    20 μM primer c (1 μM)                    --        5       --    20 μM primer d (1 μM)                    --        5       5    10 μg/μl pT2 (0.1 μg)                    10        10      --    PCR fragment AB*                    --        --      5    PCR fragment CD*                    --        --      5    2.5 U/μl Tag polymerase                      0.5       0.5     0.5    total amount    100 μl 100 μl                                      100 μl    ______________________________________     *PCR fragments AB and CD were prepared, after the PCR reaction, by     recovering 10 μl thereof from polyacrylamide gel, and dissolving it in     5 μl of TE (10 mM TrisHCl (pH 8.0), 1 mM EDTA (pH 8.0)).

In the AD fragment obtained as described above, a BssHII site (1231-1236in SEQ ID NO:1) at the upstream side and a SplI site (2249-2254 in SEQID NO:1) at the downstream side were present, so that complete digestionwas performed with these enzymes to make replacement for a correspondingregion of the plasmid pT2 (FIG. 11).

<2> Selection of inhibition-desensitized phosphoenolpyruvate carboxylase

Escherichia coli was transformed with a plasmid obtained as describedabove, and a transformed strain was cultivated to recover the plasmid toselect one cleaved by EcoRI. With respect to selected DNA, a basesequence of the region amplified by the aforementioned PCR method wasdetermined by the dideoxy method to confirm that base replacement asexactly aimed was introduced. This plasmid was designated as pT2R438C. Astrain (AJ12874) obtained by introducing this plasmid into theaforementioned Escherichia coli F15 has been deposited as FERM P-13753in National Institute of Bioscience and Human Technology of Agency ofIndustrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan; zip code 305) on Jul. 15, 1993, transferred from theoriginal deposition to international deposition based on Budapest Treatyon Jul. 11, 1994 and has been deposited as deposition number of FERMBP-4733.

The base sequence of pT2R438C is a sequence in which 1541th and 1550thnucleotides are replaced from C to T respectively in SEQ ID NO:1.

<3> Confirmation of desensitization of inhibition of mutantphosphoenolpyruvate carboxylase by aspartic acid

Sensitivity to aspartic acid was investigated for phosphoenolpyruvatecarboxylase produced by the aforementioned Escherichia coli AJ12874harboring pT2R438C. Incidentally, as described above, because theEscherichia coli F15 is deficient in phosphoenolpyruvate carboxylase,phosphoenolpyruvate carboxylase produced by AJ12874 originates from theplasmid.

Sensitivity to aspartic acid was investigated in accordance with a knownmethod (Yoshinaga, T., Izui, K. and Katsuki, H., J. Biochem., 68,747-750 (1970)). Namely, as a result of measurement of the enzymeactivity in the presence of acetyl-coenzyme A known to affect theactivity in an activity measurement system at a concentration of 1 mM or2 mM, sensitivity to aspartic acid was measured as shown in FIG. 12.

It is apparent that the wild type enzyme substantially loses itsactivity when aspartic acid is at a high concentration, while the mutantphosphoenolpyruvate carboxylase of the present invention continues tomaintain its activity.

<4> Preparation of mutant phosphoenolpyruvate carboxylase gene (II)

In order to replace a codon of 620th lysine with a codon of serine inthe phosphoenolpyruvate carboxylase gene carried on the plasmid pT2, theoverlapping Extension method (Ho, S. N., Hunt, H. D., Horton, R. M.,Pullen, J. K. and Pease, L. R., Gene, 77, 51-59 (1989)) utilizing thePCR (Polymerase Chain Reaction) method was used. Concrete procedureswere in accordance with the method described in <1>. A plasmid carryinga mutant gene constructed with aimed replacement was designated aspT2K620S. Further, an obtained mutant enzyme was designated as K620Smutant enzyme.

<5> Confirmation of desensitization of inhibition by aspartic acidconcerning mutant phosphoenolpyruvate carboxylase.

With respect to the phosphoenolpyruvic carboxylase produced by atransformant obtained by introducing the plasmid pT2K620S into theaforementioned Escherichia coli F15, sensitivity to aspartic acid wasinvestigated. Incidentally, as described above, since the Escherichiacoli F15 lacks phosphoenolpyruvate carboxylase, any phosphoenolpyruvatecarboxylase produced by the transformant originates from the plasmid.

Sensitivity to aspartic acid was investigated in accordance with a knownmethod (Yoshinaga, T., Izui, K. and Katsuki, H., J. Biochem., 68,747-750 (1970)). Namely, as a result of measurement of the enzymeactivity in the presence of acetyl-coenzyme A known to affect theactivity in an activity measurement system at a concentration of 1 mM or2 mM, sensitivity to aspartic acid was measured as shown in FIG. 13.

It is apparent that the wild enzyme substantially loses its activitywhen aspartic acid is at a high concentration, while the typephosphoenolpyruvate carboxylase of the present invention continues tomaintain its activity.

In FIG. 13, sensitivity to aspartic acid is also depicted for a mutantphosphoenolpyruvate carboxylase in which 650th lysine is replaced withalanime (K650A mutant enzyme), and for a mutant phosphoenolpyruvatecarboxylase in which 491th lysine is replaced with serine (K491A mutantenzyme). In the case of these mutant enzymes, inhibition by asparticacid was not desensitized.

INDUSTRIAL APPLICABILITY

The DNA sequence of the present invention codes for the mutantphosphoenolpyruvate carboxylase, and the microorganism harboring thisDNA sequence produces the aforementioned enzyme.

The mutant phosphoenolpyruvate carboxylase of the present invention doesnot substantially undergo activity inhibition by aspartic acid, so thatit can be utilized for fermentative production of amino acids subjectedto regulation of biosynthesis by aspartic acid and the like.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 12    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5186 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: circular    (ii) MOLECULE TYPE: DNA (genomic)    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Escherichia coli    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 237..2888    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TCGACCGGCGATTTTTTAACATTTCCATAAGTTACGCTTATTTAAAGCGTCGTGAATTTA60    ATGACGTAAATTCCTGCTATTTATTCGTTTGCTGAAGCGATTTCGCAGCATTTGACGTCA120    CCGCTTTTACGTGGCTTTATAAAAGACGACGAAAAGCAAAGCCCGAGCATATTCGCGCCA180    ATGCGACGTGAAGGATACAGGGCTATCAAACGATAAGATGGGGTGTCTGGGGTAAT236    ATGAACGAACAATATTCCGCATTGCGTAGTAATGTCAGTATGCTCGGC284    MetAsnGluGlnTyrSerAlaLeuArgSerAsnValSerMetLeuGly    151015    AAAGTGCTGGGAGAAACCATCAAGGATGCGTTGGGAGAACACATTCTT332    LysValLeuGlyGluThrIleLysAspAlaLeuGlyGluHisIleLeu    202530    GAACGCGTAGAAACTATCCGTAAGTTGTCGAAATCTTCACGCGCTGGC380    GluArgValGluThrIleArgLysLeuSerLysSerSerArgAlaGly    354045    AATGATGCTAACCGCCAGGAGTTGCTCACCACCTTACAAAATTTGTCG428    AsnAspAlaAsnArgGlnGluLeuLeuThrThrLeuGlnAsnLeuSer    505560    AACGACGAGCTGCTGCCCGTTGCGCGTGCGTTTAGTCAGTTCCTGAAC476    AsnAspGluLeuLeuProValAlaArgAlaPheSerGlnPheLeuAsn    65707580    CTGGCCAACACCGCCGAGCAATACCACAGCATTTCGCCGAAAGGCGAA524    LeuAlaAsnThrAlaGluGlnTyrHisSerIleSerProLysGlyGlu    859095    GCTGCCAGCAACCCGGAAGTGATCGCCCGCACCCTGCGTAAACTGAAA572    AlaAlaSerAsnProGluValIleAlaArgThrLeuArgLysLeuLys    100105110    AACCAGCCGGAACTGAGCGAAGACACCATCAAAAAAGCAGTGGAATCG620    AsnGlnProGluLeuSerGluAspThrIleLysLysAlaValGluSer    115120125    CTGTCGCTGGAACTGGTCCTCACGGCTCACCCAACCGAAATTACCCGT668    LeuSerLeuGluLeuValLeuThrAlaHisProThrGluIleThrArg    130135140    CGTACACTGATCCACAAAATGGTGGAAGTGAACGCCTGTTTAAAACAG716    ArgThrLeuIleHisLysMetValGluValAsnAlaCysLeuLysGln    145150155160    CTCGATAACAAAGATATCGCTGACTACGAACACAACCAGCTGATGCGT764    LeuAspAsnLysAspIleAlaAspTyrGluHisAsnGlnLeuMetArg    165170175    CGCCTGCGCCAGTTGATCGCCCAGTCATGGCATACCGATGAAATCCGT812    ArgLeuArgGlnLeuIleAlaGlnSerTrpHisThrAspGluIleArg    180185190    AAGCTGCGTCCAAGCCCGGTAGATGAAGCCAAATGGGGCTTTGCCGTA860    LysLeuArgProSerProValAspGluAlaLysTrpGlyPheAlaVal    195200205    GTGGAAAACAGCCTGTGGCAAGGCGTACCAAATTACCTGCGCGAACTG908    ValGluAsnSerLeuTrpGlnGlyValProAsnTyrLeuArgGluLeu    210215220    AACGAACAACTGGAAGAGAACCTCGGCTACAAACTGCCCGTCGAATTT956    AsnGluGlnLeuGluGluAsnLeuGlyTyrLysLeuProValGluPhe    225230235240    GTTCCGGTCCGTTTTACTTCGTGGATGGGCGGCGACCGCGACGGCAAC1004    ValProValArgPheThrSerTrpMetGlyGlyAspArgAspGlyAsn    245250255    CCGAACGTCACTGCCGATATCACCCGCCACGTCCTGCTACTCAGCCGC1052    ProAsnValThrAlaAspIleThrArgHisValLeuLeuLeuSerArg    260265270    TGGAAAGCCACCGATTTGTTCCTGAAAGATATTCAGGTGCTGGTTTCT1100    TrpLysAlaThrAspLeuPheLeuLysAspIleGlnValLeuValSer    275280285    GAACTGTCGATGGTTGAAGCGACCCCTGAACTGCTGGCGCTGGTTGGC1148    GluLeuSerMetValGluAlaThrProGluLeuLeuAlaLeuValGly    290295300    GAAGAAGGTGCCGCAGAACCGTATCGCTATCTGATGAAAAACCTGCGT1196    GluGluGlyAlaAlaGluProTyrArgTyrLeuMetLysAsnLeuArg    305310315320    TCTCGCCTGATGGCGACACAGGCATGGCTGGAAGCGCGCCTGAAAGGC1244    SerArgLeuMetAlaThrGlnAlaTrpLeuGluAlaArgLeuLysGly    325330335    GAAGAACTGCCAAAACCAGAAGGCCTGCTGACACAAAACGAAGAACTG1292    GluGluLeuProLysProGluGlyLeuLeuThrGlnAsnGluGluLeu    340345350    TGGGAACCGCTCTACGCTTGCTACCAGTCACTTCAGGCGTGTGGCATG1340    TrpGluProLeuTyrAlaCysTyrGlnSerLeuGlnAlaCysGlyMet    355360365    GGTATTATCGCCAACGGCGATCTGCTCGACACCCTGCGCCGCGTGAAA1388    GlyIleIleAlaAsnGlyAspLeuLeuAspThrLeuArgArgValLys    370375380    TGTTTCGGCGTACCGCTGGTCCGTATTGATATCCGTCAGGAGAGCACG1436    CysPheGlyValProLeuValArgIleAspIleArgGlnGluSerThr    385390395400    CGTCATACCGAAGCGCTGGGCGAGCTGACCCGCTACCTCGGTATCGGC1484    ArgHisThrGluAlaLeuGlyGluLeuThrArgTyrLeuGlyIleGly    405410415    GACTACGAAAGCTGGTCAGAGGCCGACAAACAGGCGTTCCTGATCCGC1532    AspTyrGluSerTrpSerGluAlaAspLysGlnAlaPheLeuIleArg    420425430    GAACTGAACTCCAAACGTCCGCTTCTGCCGCGCAACTGGCAACCAAGC1580    GluLeuAsnSerLysArgProLeuLeuProArgAsnTrpGlnProSer    435440445    GCCGAAACGCGCGAAGTGCTCGATACCTGCCAGGTGATTGCCGAAGCA1628    AlaGluThrArgGluValLeuAspThrCysGlnValIleAlaGluAla    450455460    CCGCAAGGCTCCATTGCCGCCTACGTGATCTCGATGGCGAAAACGCCG1676    ProGlnGlySerIleAlaAlaTyrValIleSerMetAlaLysThrPro    465470475480    TCCGACGTACTGGCTGTCCACCTGCTGCTGAAAGAAGCGGGTATCGGG1724    SerAspValLeuAlaValHisLeuLeuLeuLysGluAlaGlyIleGly    485490495    TTTGCGATGCCGGTTGCTCCGCTGTTTGAAACCCTCGATGATCTGAAC1772    PheAlaMetProValAlaProLeuPheGluThrLeuAspAspLeuAsn    500505510    AACGCCAACGATGTCATGACCCAGCTGCTCAATATTGACTGGTATCGT1820    AsnAlaAsnAspValMetThrGlnLeuLeuAsnIleAspTrpTyrArg    515520525    GGCCTGATTCAGGGCAAACAGATGGTGATGATTGGCTATTCCGACTCA1868    GlyLeuIleGlnGlyLysGlnMetValMetIleGlyTyrSerAspSer    530535540    GCAAAAGATGCGGGAGTGATGGCAGCTTCCTGGGCGCAATATCAGGCA1916    AlaLysAspAlaGlyValMetAlaAlaSerTrpAlaGlnTyrGlnAla    545550555560    CAGGATGCATTAATCAAAACCTGCGAAAAAGCGGGTATTGAGCTGACG1964    GlnAspAlaLeuIleLysThrCysGluLysAlaGlyIleGluLeuThr    565570575    TTGTTCCACGGTCGCGGCGGTTCCATTGGTCGCGGCGGCGCACCTGCT2012    LeuPheHisGlyArgGlyGlySerIleGlyArgGlyGlyAlaProAla    580585590    CATGCGGCGCTGCTGTCACAACCGCCAGGAAGCCTGAAAGGCGGCCTG2060    HisAlaAlaLeuLeuSerGlnProProGlySerLeuLysGlyGlyLeu    595600605    CGCGTAACCGAACAGGGCGAGATGATCCGCTTTAAATATGGTCTGCCA2108    ArgValThrGluGlnGlyGluMetIleArgPheLysTyrGlyLeuPro    610615620    GAAATCACCGTCAGCAGCCTGTCGCTTTATACCGGGGCGATTCTGGAA2156    GluIleThrValSerSerLeuSerLeuTyrThrGlyAlaIleLeuGlu    625630635640    GCCAACCTGCTGCCACCGCCGGAGCCGAAAGAGAGCTGGCGTCGCATT2204    AlaAsnLeuLeuProProProGluProLysGluSerTrpArgArgIle    645650655    ATGGATGAACTGTCAGTCATCTCCTGCGATGTCTACCGCGGCTACGTA2252    MetAspGluLeuSerValIleSerCysAspValTyrArgGlyTyrVal    660665670    CGTGAAAACAAAGATTTTGTGCCTTACTTCCGCTCCGCTACGCCGGAA2300    ArgGluAsnLysAspPheValProTyrPheArgSerAlaThrProGlu    675680685    CAAGAACTGGGCAAACTGCCGTTGGGTTCACGTCCGGCGAAACGTCGC2348    GlnGluLeuGlyLysLeuProLeuGlySerArgProAlaLysArgArg    690695700    CCAACCGGCGGCGTCGAGTCACTACGCGCCATTCCGTGGATCTTCGCC2396    ProThrGlyGlyValGluSerLeuArgAlaIleProTrpIlePheAla    705710715720    TGGACGCAAAACCGTCTGATGCTCCCCGCCTGGCTGGGTGCAGGTACG2444    TrpThrGlnAsnArgLeuMetLeuProAlaTrpLeuGlyAlaGlyThr    725730735    GCGCTGCAAAAAGTGGTCGAAGACGGCAAACAGAGCGAGCTGGAGGCT2492    AlaLeuGlnLysValValGluAspGlyLysGlnSerGluLeuGluAla    740745750    ATGTGCCGCGATTGGCCATTCTTCTCGACGCGTCTCGGCATGCTGGAG2540    MetCysArgAspTrpProPhePheSerThrArgLeuGlyMetLeuGlu    755760765    ATGGTCTTCGCCAAAGCAGACCTGTGGCTGGCGGAATACTATGACCAA2588    MetValPheAlaLysAlaAspLeuTrpLeuAlaGluTyrTyrAspGln    770775780    CGCCTGGTAGACAAAGCACTGTGGCCGTTAGGTAAAGAGTTACGCAAC2636    ArgLeuValAspLysAlaLeuTrpProLeuGlyLysGluLeuArgAsn    785790795800    CTGCAAGAAGAAGACATCAAAGTGGTGCTGGCGATTGCCAACGATTCC2684    LeuGlnGluGluAspIleLysValValLeuAlaIleAlaAsnAspSer    805810815    CATCTGATGGCCGATCTGCCGTGGATTGCAGAGTCTATTCAGCTACGG2732    HisLeuMetAlaAspLeuProTrpIleAlaGluSerIleGlnLeuArg    820825830    AATATTTACACCGACCCGCTGAACGTATTGCAGGCCGAGTTGCTGCAC2780    AsnIleTyrThrAspProLeuAsnValLeuGlnAlaGluLeuLeuHis    835840845    CGCTCCCGCCAGGCAGAAAAAGAAGGCCAGGAACCGGATCCTCGCGTC2828    ArgSerArgGlnAlaGluLysGluGlyGlnGluProAspProArgVal    850855860    GAACAAGCGTTAATGGTCACTATTGCCGGGATTGCGGCAGGTATGCGT2876    GluGlnAlaLeuMetValThrIleAlaGlyIleAlaAlaGlyMetArg    865870875880    AATACCGGCTAATCTTCCTCTTCTGCAAACCCTCGTGCTTTTGCGCGAGGGT2928    AsnThrGly    TTTCTGAAATACTTCTGTTCTAACACCCTCGTTTTCAATATATTTCTGTCTGCATTTTAT2988    TCAAATTCTGAATATACCTTCAGATATCCTTAAGGGCCTCGTGATACGCCTATTTTTATA3048    GGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGT3108    GCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG3168    ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACA3228    TTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCC3288    AGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACAT3348    CGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCC3408    AATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGG3468    GCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACC3528    AGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCAT3588    AACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGA3648    GCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACC3708    GGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGC3768    AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT3828    AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGC3888    TGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGC3948    AGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA4008    GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCA4068    TTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTT4128    TTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTA4188    ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG4248    AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGC4308    GGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG4368    CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAA4428    GAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGC4488    CAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGC4548    GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTA4608    CACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAG4668    AAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCT4728    TCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA4788    GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGC4848    GGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTT4908    ATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCG4968    CAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACG5028    CAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGAAGGGTTGGTTTGCGCAT5088    TCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGA5148    GGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGG5186    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 883 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetAsnGluGlnTyrSerAlaLeuArgSerAsnValSerMetLeuGly    151015    LysValLeuGlyGluThrIleLysAspAlaLeuGlyGluHisIleLeu    202530    GluArgValGluThrIleArgLysLeuSerLysSerSerArgAlaGly    354045    AsnAspAlaAsnArgGlnGluLeuLeuThrThrLeuGlnAsnLeuSer    505560    AsnAspGluLeuLeuProValAlaArgAlaPheSerGlnPheLeuAsn    65707580    LeuAlaAsnThrAlaGluGlnTyrHisSerIleSerProLysGlyGlu    859095    AlaAlaSerAsnProGluValIleAlaArgThrLeuArgLysLeuLys    100105110    AsnGlnProGluLeuSerGluAspThrIleLysLysAlaValGluSer    115120125    LeuSerLeuGluLeuValLeuThrAlaHisProThrGluIleThrArg    130135140    ArgThrLeuIleHisLysMetValGluValAsnAlaCysLeuLysGln    145150155160    LeuAspAsnLysAspIleAlaAspTyrGluHisAsnGlnLeuMetArg    165170175    ArgLeuArgGlnLeuIleAlaGlnSerTrpHisThrAspGluIleArg    180185190    LysLeuArgProSerProValAspGluAlaLysTrpGlyPheAlaVal    195200205    ValGluAsnSerLeuTrpGlnGlyValProAsnTyrLeuArgGluLeu    210215220    AsnGluGlnLeuGluGluAsnLeuGlyTyrLysLeuProValGluPhe    225230235240    ValProValArgPheThrSerTrpMetGlyGlyAspArgAspGlyAsn    245250255    ProAsnValThrAlaAspIleThrArgHisValLeuLeuLeuSerArg    260265270    TrpLysAlaThrAspLeuPheLeuLysAspIleGlnValLeuValSer    275280285    GluLeuSerMetValGluAlaThrProGluLeuLeuAlaLeuValGly    290295300    GluGluGlyAlaAlaGluProTyrArgTyrLeuMetLysAsnLeuArg    305310315320    SerArgLeuMetAlaThrGlnAlaTrpLeuGluAlaArgLeuLysGly    325330335    GluGluLeuProLysProGluGlyLeuLeuThrGlnAsnGluGluLeu    340345350    TrpGluProLeuTyrAlaCysTyrGlnSerLeuGlnAlaCysGlyMet    355360365    GlyIleIleAlaAsnGlyAspLeuLeuAspThrLeuArgArgValLys    370375380    CysPheGlyValProLeuValArgIleAspIleArgGlnGluSerThr    385390395400    ArgHisThrGluAlaLeuGlyGluLeuThrArgTyrLeuGlyIleGly    405410415    AspTyrGluSerTrpSerGluAlaAspLysGlnAlaPheLeuIleArg    420425430    GluLeuAsnSerLysArgProLeuLeuProArgAsnTrpGlnProSer    435440445    AlaGluThrArgGluValLeuAspThrCysGlnValIleAlaGluAla    450455460    ProGlnGlySerIleAlaAlaTyrValIleSerMetAlaLysThrPro    465470475480    SerAspValLeuAlaValHisLeuLeuLeuLysGluAlaGlyIleGly    485490495    PheAlaMetProValAlaProLeuPheGluThrLeuAspAspLeuAsn    500505510    AsnAlaAsnAspValMetThrGlnLeuLeuAsnIleAspTrpTyrArg    515520525    GlyLeuIleGlnGlyLysGlnMetValMetIleGlyTyrSerAspSer    530535540    AlaLysAspAlaGlyValMetAlaAlaSerTrpAlaGlnTyrGlnAla    545550555560    GlnAspAlaLeuIleLysThrCysGluLysAlaGlyIleGluLeuThr    565570575    LeuPheHisGlyArgGlyGlySerIleGlyArgGlyGlyAlaProAla    580585590    HisAlaAlaLeuLeuSerGlnProProGlySerLeuLysGlyGlyLeu    595600605    ArgValThrGluGlnGlyGluMetIleArgPheLysTyrGlyLeuPro    610615620    GluIleThrValSerSerLeuSerLeuTyrThrGlyAlaIleLeuGlu    625630635640    AlaAsnLeuLeuProProProGluProLysGluSerTrpArgArgIle    645650655    MetAspGluLeuSerValIleSerCysAspValTyrArgGlyTyrVal    660665670    ArgGluAsnLysAspPheValProTyrPheArgSerAlaThrProGlu    675680685    GlnGluLeuGlyLysLeuProLeuGlySerArgProAlaLysArgArg    690695700    ProThrGlyGlyValGluSerLeuArgAlaIleProTrpIlePheAla    705710715720    TrpThrGlnAsnArgLeuMetLeuProAlaTrpLeuGlyAlaGlyThr    725730735    AlaLeuGlnLysValValGluAspGlyLysGlnSerGluLeuGluAla    740745750    MetCysArgAspTrpProPhePheSerThrArgLeuGlyMetLeuGlu    755760765    MetValPheAlaLysAlaAspLeuTrpLeuAlaGluTyrTyrAspGln    770775780    ArgLeuValAspLysAlaLeuTrpProLeuGlyLysGluLeuArgAsn    785790795800    LeuGlnGluGluAspIleLysValValLeuAlaIleAlaAsnAspSer    805810815    HisLeuMetAlaAspLeuProTrpIleAlaGluSerIleGlnLeuArg    820825830    AsnIleTyrThrAspProLeuAsnValLeuGlnAlaGluLeuLeuHis    835840845    ArgSerArgGlnAlaGluLysGluGlyGlnGluProAspProArgVal    850855860    GluGlnAlaLeuMetValThrIleAlaGlyIleAlaAlaGlyMetArg    865870875880    AsnThrGly    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TCGCGAAGTAGCACCTGTCACTT23    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ACGGAATTCAATCTTACGGCC21    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1643 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Corynebacterium glutamicum    (B) STRAIN: ATCC13869    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 217..1482    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    TCGCGAAGTAGCACCTGTCACTTTTGTCTCAAATATTAAATCGAATATCAATATACGGTC60    TGTTTATTGGAACGCATCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGT120    GCAGAAAGAAAACACTCCTCTGGCTAGGTAGACACAGTTTATAAAGGTAGAGTTGAGCGG180    GTAACTGTCAGCACGTAGATCGAAAGGTGCACAAAGGTGGCCCTGGTCGTACAG234    MetAlaLeuValValGln    15    AAATATGGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGAAACGTC282    LysTyrGlyGlySerSerLeuGluSerAlaGluArgIleArgAsnVal    101520    GCTGAACGGATCGTTGCCACCAAGAAGGCTGGAAATGATGTCGTGGTT330    AlaGluArgIleValAlaThrLysLysAlaGlyAsnAspValValVal    253035    GTCTGCTCCGCAATGGGAGACACCACGGATGAACTTCTAGAACTTGCA378    ValCysSerAlaMetGlyAspThrThrAspGluLeuLeuGluLeuAla    404550    GCGGCAGTGAATCCCGTTCCGCCAGCTCGTGAAATGGATATGCTCCTG426    AlaAlaValAsnProValProProAlaArgGluMetAspMetLeuLeu    55606570    ACTGCTGGTGAGCGTATTTCTAACGCTCTCGTCGCCATGGCTATTGAG474    ThrAlaGlyGluArgIleSerAsnAlaLeuValAlaMetAlaIleGlu    758085    TCCCTTGGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCTGGTGTG522    SerLeuGlyAlaGluAlaGlnSerPheThrGlySerGlnAlaGlyVal    9095100    CTCACCACCGAGCGCCACGGAAACGCACGCATTGTTGACGTCACACCG570    LeuThrThrGluArgHisGlyAsnAlaArgIleValAspValThrPro    105110115    GGTCGTGTGCGTGAAGCACTCGATGAGGGCAAGATCTGCATTGTTGCT618    GlyArgValArgGluAlaLeuAspGluGlyLysIleCysIleValAla    120125130    GGTTTTCAGGGTGTTAATAAAGAAACCCGCGATGTCACCACGTTGGGT666    GlyPheGlnGlyValAsnLysGluThrArgAspValThrThrLeuGly    135140145150    CGTGGTGGTTCTGACACCACTGCAGTTGCGTTGGCAGCTGCTTTGAAC714    ArgGlyGlySerAspThrThrAlaValAlaLeuAlaAlaAlaLeuAsn    155160165    GCTGATGTGTGTGAGATTTACTCGGACGTTGACGGTGTGTATACCGCT762    AlaAspValCysGluIleTyrSerAspValAspGlyValTyrThrAla    170175180    GACCCGCGCATCGTTCCTAATGCACAGAAGCTGGAAAAGCTCAGCTTC810    AspProArgIleValProAsnAlaGlnLysLeuGluLysLeuSerPhe    185190195    GAAGAAATGCTGGAACTTGCTGCTGTTGGCTCCAAGATTTTGGTGCTG858    GluGluMetLeuGluLeuAlaAlaValGlySerLysIleLeuValLeu    200205210    CGCAGTGTTGAATACGCTCGTGCATTCAATGTGCCACTTCGCGTACGC906    ArgSerValGluTyrAlaArgAlaPheAsnValProLeuArgValArg    215220225230    TCGTCTTATAGTAATGATCCCGGCACTTTGATTGCCGGCTCTATGGAG954    SerSerTyrSerAsnAspProGlyThrLeuIleAlaGlySerMetGlu    235240245    GATATTCCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAG1002    AspIleProValGluGluAlaValLeuThrGlyValAlaThrAspLys    250255260    TCCGAAGCCAAAGTAACCGTTCTGGGTATTTCCGATAAGCCAGGCGAG1050    SerGluAlaLysValThrValLeuGlyIleSerAspLysProGlyGlu    265270275    GCTGCCAAGGTTTTCCGTGCGTTGGCTGATGCAGAAATCAACATTGAC1098    AlaAlaLysValPheArgAlaLeuAlaAspAlaGluIleAsnIleAsp    280285290    ATGGTTCTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACCGACATC1146    MetValLeuGlnAsnValSerSerValGluAspGlyThrThrAspIle    295300305310    ACGTTCACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTG1194    ThrPheThrCysProArgAlaAspGlyArgArgAlaMetGluIleLeu    315320325    AAGAAGCTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGAC1242    LysLysLeuGlnValGlnGlyAsnTrpThrAsnValLeuTyrAspAsp    330335340    CAGGTCGGCAAAGTCTCCCTCGTGGGTGCTGGCATGAAGTCTCACCCA1290    GlnValGlyLysValSerLeuValGlyAlaGlyMetLysSerHisPro    345350355    GGTGTTACCGCAGAGTTCATGGAAGCTCTGCGCGATGTCAACGTGAAC1338    GlyValThrAlaGluPheMetGluAlaLeuArgAspValAsnValAsn    360365370    ATCGAATTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTGATCCGT1386    IleGluLeuIleSerThrSerGluIleArgIleSerValLeuIleArg    375380385390    GAAGATGATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAG1434    GluAspAspLeuAspAlaAlaAlaArgAlaLeuHisGluGlnPheGln    395400405    CTGGGCGGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGCTAA1482    LeuGlyGlyGluAspGluAlaValValTyrAlaGlyThrGlyArg    410415420    AGTTTTAAAGGAGTAGTTTTACAATGACCACCATCGCAGTTGTTGGTGCAACCGGCCAGG1542    TCGGCCAGGTTATGCGCACCCTTTTGGAAGAGCGCAATTTCCCAGCTGACACTGTTCGTT1602    TCTTTGCTTCCCCGCGTTCCGCAGGCCGTAAGATTGAATTC1643    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 421 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetAlaLeuValValGlnLysTyrGlyGlySerSerLeuGluSerAla    151015    GluArgIleArgAsnValAlaGluArgIleValAlaThrLysLysAla    202530    GlyAsnAspValValValValCysSerAlaMetGlyAspThrThrAsp    354045    GluLeuLeuGluLeuAlaAlaAlaValAsnProValProProAlaArg    505560    GluMetAspMetLeuLeuThrAlaGlyGluArgIleSerAsnAlaLeu    65707580    ValAlaMetAlaIleGluSerLeuGlyAlaGluAlaGlnSerPheThr    859095    GlySerGlnAlaGlyValLeuThrThrGluArgHisGlyAsnAlaArg    100105110    IleValAspValThrProGlyArgValArgGluAlaLeuAspGluGly    115120125    LysIleCysIleValAlaGlyPheGlnGlyValAsnLysGluThrArg    130135140    AspValThrThrLeuGlyArgGlyGlySerAspThrThrAlaValAla    145150155160    LeuAlaAlaAlaLeuAsnAlaAspValCysGluIleTyrSerAspVal    165170175    AspGlyValTyrThrAlaAspProArgIleValProAsnAlaGlnLys    180185190    LeuGluLysLeuSerPheGluGluMetLeuGluLeuAlaAlaValGly    195200205    SerLysIleLeuValLeuArgSerValGluTyrAlaArgAlaPheAsn    210215220    ValProLeuArgValArgSerSerTyrSerAsnAspProGlyThrLeu    225230235240    IleAlaGlySerMetGluAspIleProValGluGluAlaValLeuThr    245250255    GlyValAlaThrAspLysSerGluAlaLysValThrValLeuGlyIle    260265270    SerAspLysProGlyGluAlaAlaLysValPheArgAlaLeuAlaAsp    275280285    AlaGluIleAsnIleAspMetValLeuGlnAsnValSerSerValGlu    290295300    AspGlyThrThrAspIleThrPheThrCysProArgAlaAspGlyArg    305310315320    ArgAlaMetGluIleLeuLysLysLeuGlnValGlnGlyAsnTrpThr    325330335    AsnValLeuTyrAspAspGlnValGlyLysValSerLeuValGlyAla    340345350    GlyMetLysSerHisProGlyValThrAlaGluPheMetGluAlaLeu    355360365    ArgAspValAsnValAsnIleGluLeuIleSerThrSerGluIleArg    370375380    IleSerValLeuIleArgGluAspAspLeuAspAlaAlaAlaArgAla    385390395400    LeuHisGluGlnPheGlnLeuGlyGlyGluAspGluAlaValValTyr    405410415    AlaGlyThrGlyArg    420    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1643 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Corynebacterium glutamicum    (B) STRAIN: ATCC13869    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 964..1482    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    TCGCGAAGTAGCACCTGTCACTTTTGTCTCAAATATTAAATCGAATATCAATATACGGTC60    TGTTTATTGGAACGCATCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGT120    GCAGAAAGAAAACACTCCTCTGGCTAGGTAGACACAGTTTATAAAGGTAGAGTTGAGCGG180    GTAACTGTCAGCACGTAGATCGAAAGGTGCACAAAGGTGGCCCTGGTCGTACAGAAATAT240    GGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGAAACGTCGCTGAACGGATCGTTGCC300    ACCAAGAAGGCTGGAAATGATGTCGTGGTTGTCTGCTCCGCAATGGGAGACACCACGGAT360    GAACTTCTAGAACTTGCAGCGGCAGTGAATCCCGTTCCGCCAGCTCGTGAAATGGATATG420    CTCCTGACTGCTGGTGAGCGTATTTCTAACGCTCTCGTCGCCATGGCTATTGAGTCCCTT480    GGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCTGGTGTGCTCACCACCGAGCGCCAC540    GGAAACGCACGCATTGTTGACGTCACACCGGGTCGTGTGCGTGAAGCACTCGATGAGGGC600    AAGATCTGCATTGTTGCTGGTTTTCAGGGTGTTAATAAAGAAACCCGCGATGTCACCACG660    TTGGGTCGTGGTGGTTCTGACACCACTGCAGTTGCGTTGGCAGCTGCTTTGAACGCTGAT720    GTGTGTGAGATTTACTCGGACGTTGACGGTGTGTATACCGCTGACCCGCGCATCGTTCCT780    AATGCACAGAAGCTGGAAAAGCTCAGCTTCGAAGAAATGCTGGAACTTGCTGCTGTTGGC840    TCCAAGATTTTGGTGCTGCGCAGTGTTGAATACGCTCGTGCATTCAATGTGCCACTTCGC900    GTACGCTCGTCTTATAGTAATGATCCCGGCACTTTGATTGCCGGCTCTATGGAGGATATT960    CCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAGTCCGAA1008    MetGluGluAlaValLeuThrGlyValAlaThrAspLysSerGlu    151015    GCCAAAGTAACCGTTCTGGGTATTTCCGATAAGCCAGGCGAGGCTGCC1056    AlaLysValThrValLeuGlyIleSerAspLysProGlyGluAlaAla    202530    AAGGTTTTCCGTGCGTTGGCTGATGCAGAAATCAACATTGACATGGTT1104    LysValPheArgAlaLeuAlaAspAlaGluIleAsnIleAspMetVal    354045    CTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACCGACATCACGTTC1152    LeuGlnAsnValSerSerValGluAspGlyThrThrAspIleThrPhe    505560    ACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTGAAGAAG1200    ThrCysProArgAlaAspGlyArgArgAlaMetGluIleLeuLysLys    657075    CTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGACCAGGTC1248    LeuGlnValGlnGlyAsnTrpThrAsnValLeuTyrAspAspGlnVal    80859095    GGCAAAGTCTCCCTCGTGGGTGCTGGCATGAAGTCTCACCCAGGTGTT1296    GlyLysValSerLeuValGlyAlaGlyMetLysSerHisProGlyVal    100105110    ACCGCAGAGTTCATGGAAGCTCTGCGCGATGTCAACGTGAACATCGAA1344    ThrAlaGluPheMetGluAlaLeuArgAspValAsnValAsnIleGlu    115120125    TTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTGATCCGTGAAGAT1392    LeuIleSerThrSerGluIleArgIleSerValLeuIleArgGluAsp    130135140    GATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAGCTGGGC1440    AspLeuAspAlaAlaAlaArgAlaLeuHisGluGlnPheGlnLeuGly    145150155    GGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGCTAA1482    GlyGluAspGluAlaValValTyrAlaGlyThrGlyArg    160165170    AGTTTTAAAGGAGTAGTTTTACAATGACCACCATCGCAGTTGTTGGTGCAACCGGCCAGG1542    TCGGCCAGGTTATGCGCACCCTTTTGGAAGAGCGCAATTTCCCAGCTGACACTGTTCGTT1602    TCTTTGCTTCCCCGCGTTCCGCAGGCCGTAAGATTGAATTC1643    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 172 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    ValGluGluAlaValLeuThrGlyValAlaThrAspLysSerGluAla    151015    LysValThrValLeuGlyIleSerAspLysProGlyGluAlaAlaLys    202530    ValPheArgAlaLeuAlaAspAlaGluIleAsnIleAspMetValLeu    354045    GlnAsnValSerSerValGluAspGlyThrThrAspIleThrPheThr    505560    CysProArgAlaAspGlyArgArgAlaMetGluIleLeuLysLysLeu    65707580    GlnValGlnGlyAsnTrpThrAsnValLeuTyrAspAspGlnValGly    859095    LysValSerLeuValGlyAlaGlyMetLysSerHisProGlyValThr    100105110    AlaGluPheMetGluAlaLeuArgAspValAsnValAsnIleGluLeu    115120125    IleSerThrSerGluIleArgIleSerValLeuIleArgGluAspAsp    130135140    LeuAspAlaAlaAlaArgAlaLeuHisGluGlnPheGlnLeuGlyGly    145150155160    GluAspGluAlaValValTyrAlaGlyThrGlyArg    165170    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    AAAAACCTGCGTTCTC16    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    TGACTTAAGGTTTACAGGCC20    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    ACTGAATTCCAAATGTCCGC20    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    AAGTGCAGGCCGTTT15    __________________________________________________________________________

What is claimed is:
 1. A method of producing an amino acid,comprising:selecting a microorganism of the genus Escherichia containinga DNA sequence encoding a mutant phosphoenolpyruvate carboxylasedesensitized to feedback inhibition by aspartic acid by growingEscherichia microorganisms in the presence of a wild-typephosphoenolpyruvate carboxylase inhibitor selected from the groupconsisting of 3-bromopyruvate, aspartic acid-β-hydrazide andDL-threo-β-hydroxyaspartic acid; culturing a microorganism of the genusEscherichia or coryneform bacteria transformed with the DNA sequenceencoding a mutant phosphoenolpyruvate carboxylase in a suitable medium;and separating from the medium an amino acid selected from the groupconsisting of L-lysine, L-threonine, L-methionine, L-isoleucine,L-glutamic acid, L-arginine and L-proline.
 2. The method of claim 1,wherein the mutant phosphoenolpyruvate carboxylase has the glutamic acidresidue at the 625^(th) position from the N-terminus mutated to an aminoacid residue other than glutamic acid.
 3. A method according to claim 1,wherein the mutant phosphoenolpyruvate carboxylase has the glutamic acidresidue at the 625^(th) position from the N-terminus replaced withlysine.
 4. A method according to claim 1, wherein the mutantphosphoenolpyruvate carboxylase has the arginine residue at the 222^(nd)position mutated to an amino acid residue other than arginine and theglutamic acid residue at the 223^(rd) position mutated to an amino acidresidue other than glutamic acid, wherein the amino acid positions aremeasured from the N-terminus of the phosphoenolpyruvate carboxylase. 5.A method according to claim 1, wherein the mutant phosphoenolpyruvatecarboxylase has the arginine residue at the 222^(nd) position replacedwith histidine and the glutamic acid residue at the 223^(rd) positionreplaced with lysine, wherein the amino acid positions are measured fromthe N-terminus of the phosphoenolpyruvate carboxylase.
 6. A methodaccording to claim 1, wherein the mutant phosphoenolpyruvate carboxylasehas the serine residue at the 288^(th) position mutated to an amino acidresidue other than serine, the glutamic acid residue at the 289^(th)position mutated to an amino acid residue other than glutamic acid, themethionine residue at the 551^(st) position mutated to an amino acidother than methionine, and the glutamic acid residue at the 804^(th)position mutated to an amino acid other than glutamic acid, wherein theamino acid positions are measured from the N-terminus of thephosphoenolpyruvate carboxylase.
 7. A method according to claim 1,wherein the mutant phosphoenolpyruvate carboxylase has the serineresidue at the 288^(th) position replaced with phenylalanine, theglutamic acid residue at the 289^(th) position replaced with lysine, themethionine residue at the 551^(st) position replaced with isoleucine,and the glutamic acid residue at the 804^(th) position replaced withlysine, wherein the amino acid positions are measured from theN-terminus of the phosphoenolpyruvate carboxylase.
 8. A method accordingto claim 1, wherein the mutant phosphoenolpyruvate carboxylase has thealanine residue at the 867^(th) position as measured from the N-terminusmutated to an amino acid residue other than alanine.
 9. A methodaccording to claim 1, wherein the mutant phosphoenolpyruvate carboxylasehas the alanine residue at the 867^(th) position as measured from theN-terminus replaced with threonine.
 10. A method of producing aminoacid, comprising:cultivating a microorganism belonging to the genusEscherichia or coryneform bacteria in a suitable medium; and separating,from the medium, an amino acid selected from the group consisting ofL-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid,L-arginine and L-proline, wherein the microorganism is transformed byallowing a DNA fragment to be integrated in chromosomal DNA ortransformed with a recombinant DNA formed by ligating the DNA fragmentwith a vector DNA capable of autonomously replication in cells ofbacteria belonging to the genus Escherichia or coryneform bacteria,wherein the DNA fragment encodes a mutant phosphoenolpyruvatecarboxylase originating from a microorganism belonging to the genusEscherichia; the mutant phosphoenolpyruvate carboxylase has mutation todesensitize feedback inhibition of the phosphoenolpyruvate carboxylaseby aspartic acid; and the mutant phosphoenolpyruvate carboxylase has thearginine residue at the 438^(th) position from the N-terminus mutated toan amino acid residue other than arginine.
 11. A method according toclaim 10, wherein the mutant phosphoenolpyruvate carboxylase has thearginine residue at the 438^(th) position replaced with cysteine.
 12. Amethod of producing amino acid, comprising:cultivating a microorganismbelonging to the genus Escherichia or coryneform bacteria in a suitablemedium; and separating, from the medium, an amino acid selected from thegroup consisting of L-lysine, L-threonine, L-methionine, L-isoleucine,L-glutamic acid, L-arginine and L-proline, wherein the microorganism istransformed by allowing a DNA fragment to be integrated in chromosomalDNA or transformed with a recombinant DNA formed by ligating the DNAfragment with a vector DNA capable of autonomously replication in cellsof bacteria belonging to the genus Escherichia or coryneform bacteria;the DNA fragment encodes a mutant phosphoenolpyruvate carboxylaseoriginating from a microorganism belonging to the genus Escherichia; themutant phosphoenolpyruvate carboxylase has mutation to desensitizefeedback inhibition of the phosphoenolpyruvate carboxylase by asparticacid; and the mutant phosphoenolpyruvate carboxylase has the lysineresidue at the 620^(th) position from the N-terminus mutated to an aminoacid residue other than lysine.
 13. A method according to claim 12,wherein the mutant phosphoenolpyruvate carboxylase has the lysineresidue at the 620^(th) position replaced with serine.